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Sample records for sphingolipids

  1. Sphingolipids in parasitic protozoa

    PubMed Central

    Zhang, Kai; Bangs, James D.; Beverley, Stephen M.

    2009-01-01

    The surface of most protozoan parasites relies heavily upon lipid-anchored molecules, to form protective barriers and play critical functions required for infectivity. Sphingolipids (SLs) play important roles through their abundance and involvement in membrane microdomain formation, as well as serving as the lipid anchor for many of these molecules, and in some but possibly not all species, as important signaling molecules. Interactions of parasite sphingolipid metabolism with that of the host may potentially contribute to parasite survival and/or host defense. In this chapter we summarize current knowledge of SL structure, synthesis and function in several of the major parasitic protozoan groups. PMID:20919659

  2. Novel drugs targeting sphingolipid metabolism.

    PubMed

    Bhabak, Krishna P; Arenz, Christoph

    2013-01-01

    While the evidence for an involvement of sphingolipids (SLs) in a variety of diseases is rapidly increasing, the development of sphingolipid-related drugs is still in its infancy. In fact, the recently FDA-approved fingolimod or FTY-720 (see chapter by J. Pfeilschifter for more information) is the first drug on the market to interfere with sphingolipid signaling. The reasons for this lagging are manifold and within this chapter we try to name some of them. Ceramide is in the center of sphingolipid metabolism. We describe the most important and most recent inhibitors for enzymes controlling cellular ceramide levels.

  3. The Yeast Sphingolipid Signaling Landscape

    PubMed Central

    Montefusco, David J.; Matmati, Nabil

    2014-01-01

    Sphingolipids are recognized as signaling mediators in a growing number of pathways, and represent potential targets to address many diseases. The study of sphingolipid signaling in yeast has created a number of breakthroughs in the field, and has the potential to lead future advances. The aim of this article is to provide an inclusive view of two major frontiers in yeast sphingolipid signaling. In the first section, several key studies in the field of sphingolipidomics are consolidated to create a yeast sphingolipidome that ranks nearly all known sphingolipid species by their level in a resting yeast cell. The second section presents an overview of most known phenotypes identified for sphingolipid gene mutants, presented with the intention of illuminating not yet discovered connections outside and inside of the field. PMID:24220500

  4. Sphingolipids in spinal cord injury

    PubMed Central

    Jones, Zachary B; Ren, Yi

    2016-01-01

    Spinal cord injury (SCI) is a debilitating condition that affects millions of individuals worldwide. Despite progress over the last few decades, the molecular mechanisms of secondary SCI that continue to occur days and weeks after the original trauma remain poorly understood. As a result, current therapies for SCI are only marginally effective. Sphingolipids, a diverse class of bioactive lipids, have been shown to regulate SCI repair and key secondary injury processes such as apoptosis, ischemia and inflammation. This review will discuss the numerous roles of sphingolipids and highlight the potential of sphingolipid-targeted therapies for SCI. PMID:27570580

  5. Molecular facets of sphingolipids: mediators of diseases.

    PubMed

    Ozbayraktar, Fatma Betul Kavun; Ulgen, Kutlu O

    2009-07-01

    Sphingolipids constitute a biologically active lipid class that is significantly important from both structural and regulatory aspects. The manipulation of sphingolipid metabolism is currently being studied as a novel strategy for cancer therapy. The basics of this therapeutic approach lie in the regulation property of sphingolipids on cellular processes, which are important in a cell's fate, such as cell proliferation, apoptosis, cell cycle arrest, senescence, and inflammation. Furthermore, the mutations in the enzymes catalyzing some specific reactions in the sphingolipid metabolism cause mortal lysosomal storage diseases like Fabry, Gaucher, Niemann-Pick, Farber, Krabbe, and Metachromatic Leukodystrophy. Therefore, the alteration of the sphingolipid metabolic pathway determines the choice between life and death. Understanding the sphingolipid metabolism and regulation is significant for the development of new therapeutic approaches for all sphingolipid-related diseases, as well as for cancer. An important feature of the sphingolipid metabolic pathway is the compartmentalization into endoplasmic reticulum, the Golgi apparatus, lysosome and plasma membrane, and this compartmentalization makes the transport of sphingolipids critical for proper functioning. This paper focuses on the structures, metabolic pathways, localization, transport mechanisms, and diseases of sphingolipids in Saccharomyces cerevisiae and humans, and provides the latest comprehensive information on sphingolipid research.

  6. Sphingolipids and response to chemotherapy.

    PubMed

    Dimanche-Boitrel, Marie-Thérèse; Rebillard, Amélie

    2013-01-01

    Chemotherapy is frequently used to treat primary or metastatic cancers, but intrinsic or acquired drug resistance limits its efficiency. Sphingolipids are important regulators of various cellular processes including proliferation, apoptosis, differentiation, angiogenesis, stress, and inflammatory responses which are linked to various aspects of cancer, like tumor growth, neoangiogenesis, and response to chemotherapy. Ceramide, the central molecule of sphingolipid metabolism, generally mediates antiproliferative and proapoptotic functions, whereas sphingosine-1-phosphate and other derivatives have opposing effects. Among the variety of enzymes that control ceramide generation, acid or neutral sphingomyelinases and ceramide synthases are important targets to allow killing of cancer cells by chemotherapeutic drugs. On the contrary, glucosylceramide synthase, ceramidase, and sphingosine kinase are other targets driving cancer cell resistance to chemotherapy. This chapter focuses on ceramide-based mechanisms leading to cancer therapy sensitization or resistance which could have some impacts on the development of novel cancer therapeutic strategies.

  7. Distribution and functions of sterols and sphingolipids.

    PubMed

    Hannich, J Thomas; Umebayashi, Kyohei; Riezman, Howard

    2011-05-01

    Sterols and sphingolipids are considered mainly eukaryotic lipids even though both are present in some prokaryotes, with sphingolipids being more widespread than sterols. Both sterols and sphingolipids differ in their structural features in vertebrates, plants, and fungi. Interestingly, some invertebrates cannot synthesize sterols de novo and seem to have a reduced dependence on sterols. Sphingolipids and sterols are found in the plasma membrane, but we do not have a clear picture of their precise intracellular localization. Advances in lipidomics and subcellular fractionation should help to improve this situation. Genetic approaches have provided insights into the diversity of sterol and sphingolipid functions in eukaryotes providing evidence that these two lipid classes function together. Intermediates in sphingolipid biosynthesis and degradation are involved in signaling pathways, whereas sterol structures are converted to hormones. Both lipids have been implicated in regulating membrane trafficking.

  8. Distribution and Functions of Sterols and Sphingolipids

    PubMed Central

    Hannich, J. Thomas; Umebayashi, Kyohei; Riezman, Howard

    2011-01-01

    Sterols and sphingolipids are considered mainly eukaryotic lipids even though both are present in some prokaryotes, with sphingolipids being more widespread than sterols. Both sterols and sphingolipids differ in their structural features in vertebrates, plants, and fungi. Interestingly, some invertebrates cannot synthesize sterols de novo and seem to have a reduced dependence on sterols. Sphingolipids and sterols are found in the plasma membrane, but we do not have a clear picture of their precise intracellular localization. Advances in lipidomics and subcellular fractionation should help to improve this situation. Genetic approaches have provided insights into the diversity of sterol and sphingolipid functions in eukaryotes providing evidence that these two lipid classes function together. Intermediates in sphingolipid biosynthesis and degradation are involved in signaling pathways, whereas sterol structures are converted to hormones. Both lipids have been implicated in regulating membrane trafficking. PMID:21454248

  9. Sphingolipids in the DNA damage response.

    PubMed

    Carroll, Brittany; Donaldson, Jane Catalina; Obeid, Lina

    2015-05-01

    Recently, sphingolipid metabolizing enzymes have emerged as important targets of many chemotherapeutics and DNA damaging agents and therefore play significant roles in mediating the physiological response of the cell to DNA damage. In this review we will highlight points of connection between the DNA damage response (DDR) and sphingolipid metabolism; specifically how certain sphingolipid enzymes are regulated in response to DNA damage and how the bioactive lipids produced by these enzymes affect cell fate. Published by Elsevier Ltd.

  10. Sphingolipids in the DNA Damage Response

    PubMed Central

    Carroll, Brittany; Donaldson, Cat; Obeid, Lina

    2014-01-01

    Recently, sphingolipid metabolizing enzymes have emerged as important targets of many chemotherapeutics and DNA damaging agents and therefore play significant roles in mediating the physiological response of the cell to DNA damage. In this review we will highlight points of connection between the DNA damage response (DDR) and sphingolipid metabolism; specifically how certain sphingolipid enzymes are regulated in response to DNA damage and how the bioactive lipids produced by these enzymes affect cell fate. PMID:25434743

  11. Sphingolipids in inflammation: roles and implications.

    PubMed

    Pettus, B J; Chalfant, C E; Hannun, Y A

    2004-06-01

    Sphingolipids, historically described as potential reservoirs for bioactive lipids, presently define a new family of cellular mediators, joining the well-established glycerolipid-derived mediators of signal transduction such as diacylglycerol, phosphatidylinositides, and eicosanoids. Sphingolipid metabolism is clearly involved in the regulation of cell growth, differentiation, and programmed cell death. Indeed, a majority of the greater than four thousand studies conducted on sphingolipids during the past five years were investigations of the role of sphingolipids as cellular bioregulators. Studies spanning more than a decade have shown multiple interactions and intersections of the sphingolipid-mediated pathways and the eicosanoid pathway. This review will discuss the emerging mechanisms by which sphingolipids induce inflammatory responses via the eicosanoid pathway in addition to linking previous literature on sphingolipids and inflammation with newer findings of distinct roles for sphingosine-1-phosphate in regulating cyclooygenase-2 and ceramide-1-phosphate in the regulation of cytosolic phospholipase A2alpha. Finally, the relationship between bioactive sphingolipids and inflammation is discussed.

  12. Separation and identification of major plant sphingolipid classes from leaves.

    PubMed

    Markham, Jonathan E; Li, Jia; Cahoon, Edgar B; Jaworski, Jan G

    2006-08-11

    Sphingolipids are major components of the plasma membrane, tonoplast, and other endomembranes of plant cells. Previous compositional analyses have focused only on individual sphingolipid classes because of the widely differing polarities of plant sphingolipids. Consequently, the total content of sphingolipid classes in plants has yet to be quantified. In addition, the major polar sphingolipid class in the model plant Arabidopsis thaliana has not been previously determined. In this report, we describe the separation and quantification of sphingolipid classes from A. thaliana leaves using hydrolysis of sphingolipids and high performance liquid chromatography (HPLC) analysis of o-phthaldialdehyde derivatives of the released long-chain bases to monitor the separation steps. An extraction solvent that contained substantial proportions of water was used to solubilized >95% of the sphingolipids from leaves. Neutral and charged sphingolipids were then partitioned by anion exchange solid phase extraction. HPLC analysis of the charged lipid fraction from A. thaliana revealed only one major anionic sphingolipid class, which was identified by mass spectrometry as hexose-hexuronic-inositolphosphoceramide. The neutral sphingolipids were predominantly composed of monohexosylceramide with lesser amounts of ceramides. Extraction and separation of sphingolipids from soybean and tomato showed that, like A. thaliana, the neutral sphingolipids consisted of ceramide and monohexosylceramides; however, the major polar sphingolipid was found to be N-acetyl-hexosamine-hexuronic-inositolphosphoceramide. In extracts from A. thaliana leaves, hexosehexuronic-inositolphosphoceramides, monohexosylceramides, and ceramides accounted for approximately 64, 34, and 2% of the total sphingolipids, respectively, suggesting an important role for the anionic sphingolipids in plant membranes.

  13. Sphingolipids inhibit vimentin-dependent cell migration.

    PubMed

    Hyder, Claire L; Kemppainen, Kati; Isoniemi, Kimmo O; Imanishi, Susumu Y; Goto, Hidemasa; Inagaki, Masaki; Fazeli, Elnaz; Eriksson, John E; Törnquist, Kid

    2015-06-01

    The sphingolipids, sphingosine 1-phosphate (S1P) and sphingosylphosphorylcholine (SPC), can induce or inhibit cellular migration. The intermediate filament protein vimentin is an inducer of migration and a marker for epithelial-mesenchymal transition. Given that keratin intermediate filaments are regulated by SPC, with consequences for cell motility, we wanted to determine whether vimentin is also regulated by sphingolipid signalling and whether it is a determinant for sphingolipid-mediated functions. In cancer cells where S1P and SPC inhibited migration, we observed that S1P and SPC induced phosphorylation of vimentin on S71, leading to a corresponding reorganization of vimentin filaments. These effects were sphingolipid-signalling-dependent, because inhibition of either the S1P2 receptor (also known as S1PR2) or its downstream effector Rho-associated kinase (ROCK, for which there are two isoforms ROCK1 and ROCK2) nullified the sphingolipid-induced effects on vimentin organization and S71 phosphorylation. Furthermore, the anti-migratory effect of S1P and SPC could be prevented by expressing S71-phosphorylation-deficient vimentin. In addition, we demonstrated, by using wild-type and vimentin-knockout mouse embryonic fibroblasts, that the sphingolipid-mediated inhibition of migration is dependent on vimentin. These results imply that this newly discovered sphingolipid-vimentin signalling axis exerts brake-and-throttle functions in the regulation of cell migration.

  14. Sphingolipid and Ceramide Homeostasis: Potential Therapeutic Targets

    PubMed Central

    Young, Simon A.; Mina, John G.; Denny, Paul W.; Smith, Terry K.

    2012-01-01

    Sphingolipids are ubiquitous in eukaryotic cells where they have been attributed a plethora of functions from the formation of structural domains to polarized cellular trafficking and signal transduction. Recent research has identified and characterised many of the key enzymes involved in sphingolipid metabolism and this has led to a heightened interest in the possibility of targeting these processes for therapies against cancers, Alzheimer's disease, and numerous important human pathogens. In this paper we outline the major pathways in eukaryotic sphingolipid metabolism and discuss these in relation to disease and therapy for both chronic and infectious conditions. PMID:22400113

  15. Monogenic neurological disorders of sphingolipid metabolism.

    PubMed

    Sabourdy, Frédérique; Astudillo, Leonardo; Colacios, Céline; Dubot, Patricia; Mrad, Marguerite; Ségui, Bruno; Andrieu-Abadie, Nathalie; Levade, Thierry

    2015-08-01

    Sphingolipids comprise a wide variety of molecules containing a sphingoid long-chain base that can be N-acylated. These lipids are particularly abundant in the central nervous system, being membrane components of neurons as well as non-neuronal cells. Direct evidence that these brain lipids play critical functions in brain physiology is illustrated by the dramatic consequences of genetic disturbances of their metabolism. Inherited defects of both synthesis and catabolism of sphingolipids are now identified in humans. These monogenic disorders are due to mutations in the genes encoding for the enzymes that catalyze either the formation or degradation of simple sphingolipids such as ceramides, or complex sphingolipids like glycolipids. They cause varying degrees of central nervous system dysfunction, quite similarly to the neurological disorders induced in mice by gene disruption of the corresponding enzymes. Herein, the enzyme deficiencies and metabolic alterations that underlie these diseases are reviewed. Their possible pathophysiological mechanisms and the functions played by sphingolipids one can deduce from these conditions are discussed. This article is part of a Special Issue entitled Brain Lipids.

  16. Developmentally Regulated Sphingolipid Synthesis in African Trypanosomes

    PubMed Central

    Sutterwala, Shaheen S.; Hsu, Fong Fu; Sevova, Elitza S.; Schwartz, Kevin J.; Zhang, Kai; Key, Phillip; Turk, John; Beverley, Stephen M.; Bangs, James D.

    2008-01-01

    Sphingolipids are essential components of eukaryotic membranes, and many unicellular eukaryotes, including kinetoplastid protozoa, are thought to synthesize exclusively inositol phosphorylceramide (IPC). Here we characterize sphingolipids from Trypanosoma brucei, and a trypanosome sphingolipid synthase gene family (TbSLS1-4) that is orthologous to Leishmania IPC synthase. Procyclic trypanosomes contain IPC, but also sphingomyelin, while surprisingly bloodstream stage parasites contain sphingomyelin and ethanolamine phosphorylceramide (EPC), but no detectable IPC. In vivo fluorescent ceramide labeling confirmed stage specific biosynthesis of both sphingomyelin and IPC. Expression of TbSLS4 in Leishmania resulted in production of sphingomyelin and EPC suggesting that the TbSLS gene family has bi-functional synthase activity. RNAi silencing of TbSLS1-4 in bloodstream trypanosomes led to rapid growth arrest and eventual cell death. Ceramide levels were increased >3-fold by silencing suggesting a toxic downstream effect mediated by this potent intracellular messenger. Topology predictions support a revised six transmembrane domain model for the kinetoplastid sphingolipid synthases consistent with the proposed mammalian SM synthase structure. This work reveals novel diversity and regulation in sphingolipid metabolism in this important group of human parasites. PMID:18699867

  17. Pleiotropic effects of sphingolipids in skeletal muscle.

    PubMed

    Bruni, P; Donati, C

    2008-11-01

    Studies of the last two decades have demonstrated that sphingolipids are important signalling molecules exerting key roles in the control of fundamental biological processes including proliferation, differentiation, motility and survival. Here we review the role of bioactive sphingolipids such as ceramide, sphingosine, sphingosine 1-phosphate, ganglioside GM3, in the regulation of skeletal muscle biology. The emerging picture is in favour of a complex role of these molecules, which appear implicated in the activation of muscle resident stem cells, their proliferation and differentiation, finalized at skeletal muscle regeneration. Moreover, they are involved in the regulation of contractile properties, tissue responsiveness to insulin and muscle fiber trophism. Hopefully, this article will provide a framework for future investigation into the field, aimed at establishing whether altered sphingolipid metabolism is implicated in the onset of skeletal muscle diseases and identifying new pharmacological targets for the therapy of multiple illnesses, including muscular dystrophies and diabetes.

  18. Plasma Sphingolipids Associated with Chronic Obstructive Pulmonary Disease Phenotypes

    PubMed Central

    Jacobson, Sean; Cruickshank, Charmion; Hughes, Grant J.; Siska, Charlotte; Ory, Daniel S.; Petrache, Irina; Schaffer, Jean E.; Reisdorph, Nichole; Kechris, Katerina

    2015-01-01

    Rationale: Chronic obstructive pulmonary disease (COPD) occurs in a minority of smokers and is characterized by intermittent exacerbations and clinical subphenotypes such as emphysema and chronic bronchitis. Although sphingolipids as a class are implicated in the pathogenesis of COPD, the particular sphingolipid species associated with COPD subphenotypes remain unknown. Objectives: To use mass spectrometry to determine which plasma sphingolipids are associated with subphenotypes of COPD. Methods: One hundred twenty-nine current and former smokers from the COPDGene cohort had 69 distinct sphingolipid species detected in plasma by targeted mass spectrometry. Of these, 23 were also measured in 131 plasma samples (117 independent subjects) using an untargeted platform in an independent laboratory. Regression analysis with adjustment for clinical covariates, correction for false discovery rate, and metaanalysis were used to test associations between COPD subphenotypes and sphingolipids. Peripheral blood mononuclear cells were used to test associations between sphingolipid gene expression and plasma sphingolipids. Measurements and Main Results: Of the measured plasma sphingolipids, five sphingomyelins were associated with emphysema; four trihexosylceramides and three dihexosylceramides were associated with COPD exacerbations. Three sphingolipids were strongly associated with sphingolipid gene expression, and 15 sphingolipid gene/metabolite pairs were differentially regulated between COPD cases and control subjects. Conclusions: There is evidence of systemic dysregulation of sphingolipid metabolism in patients with COPD. Subphenotyping suggests that sphingomyelins are strongly associated with emphysema and glycosphingolipids are associated with COPD exacerbations. PMID:25494452

  19. Sphingolipids in High Fat Diet and Obesity-Related Diseases

    PubMed Central

    Choi, Songhwa; Snider, Ashley J.

    2015-01-01

    Nutrient oversupply associated with a high fat diet (HFD) significantly alters cellular metabolism, and specifically including sphingolipid metabolism. Sphingolipids are emerging as bioactive lipids that play key roles in regulating functions, in addition to their traditional roles as membrane structure. HFD enhances de novo sphingolipid synthesis and turnover of sphingolipids via the salvage pathway, resulting in the generation of ceramide, and more specifically long chain ceramide species. Additionally, HFD elevates sphingomyelin and sphingosine-1 phosphate (S1P) levels in several tissues including liver, skeletal muscle, adipose tissue, and cardiovascular tissues. HFD-stimulated sphingolipid generation contributes to systemic insulin resistance, dysregulated lipid accumulation, and cytokine expression and secretion from skeletal muscle and adipose tissues, exacerbating obesity-related conditions. Furthermore, altered sphingolipid levels, particularly ceramide and sphingomyelin, are involved in obesity-induced endothelial dysfunction and atherosclerosis. In this review, HFD-mediated sphingolipid metabolism and its impact on HFD-induced biology and pathobiology will be discussed. PMID:26648664

  20. Plant sphingolipids: decoding the enigma of the Sphinx

    PubMed Central

    Pata, Mickael O.; Hannun, Yusuf A.; Ng, Carl K.-Y.

    2009-01-01

    Summary Sphingolipids are a ubiquitous class of lipids present in a variety of organisms including eukaryotes and bacteria. In the last two decades, research has focused on characterizing the individual species of this complex family of lipids, leading to a new field of research called sphingolipidomics. There are at least 500 (and perhaps thousands) different molecular species of sphingolipids in cells, and in Arabidopsis alone, it has been reported that there are at least 168 different sphingolipids. Plant sphingolipids can be divided into four classes: glycosyl inositol phosphoceramides (GIPCs), glycosylceramides, ceramides, and free long chain bases (LCBs). Numerous enzymes involved in plant sphingolipid metabolism have now been cloned and characterized, and, in general, there is broad conservation in the way sphingolipids are metabolized in animals, yeast and plants. Here, we review the diversity of sphingolipids reported in the literature, some of the recent advances in our understanding of sphingolipid metabolism in plants, and the physiological roles that sphingolipids and sphingolipid metabolites play in plant physiology. PMID:20028469

  1. Fostering Inflammatory Bowel Disease: Sphingolipid Strategies to Join Forces.

    PubMed

    Abdel Hadi, Loubna; Di Vito, Clara; Riboni, Laura

    2016-01-01

    Complex sphingolipids are essential structural components of intestinal membranes, providing protection and integrity to the intestinal mucosa and regulating intestinal absorption processes. The role of sphingolipid signaling has been established in numerous cellular events, including intestinal cell survival, growth, differentiation, and apoptosis. A significant body of knowledge demonstrates that intestinal sphingolipids play a crucial role, as such and through their signaling pathways, in immunity and inflammatory disorders. In this review, we report on and discuss the current knowledge on the metabolism, signaling, and functional implications of sphingolipids in inflammatory bowel disease (IBD), focusing on the different aspects of sphingolipid actions on inflammatory responses and on the potential of sphingolipid-targeted molecules as anti-IBD therapeutic agents.

  2. Fostering Inflammatory Bowel Disease: Sphingolipid Strategies to Join Forces

    PubMed Central

    Abdel Hadi, Loubna; Di Vito, Clara; Riboni, Laura

    2016-01-01

    Complex sphingolipids are essential structural components of intestinal membranes, providing protection and integrity to the intestinal mucosa and regulating intestinal absorption processes. The role of sphingolipid signaling has been established in numerous cellular events, including intestinal cell survival, growth, differentiation, and apoptosis. A significant body of knowledge demonstrates that intestinal sphingolipids play a crucial role, as such and through their signaling pathways, in immunity and inflammatory disorders. In this review, we report on and discuss the current knowledge on the metabolism, signaling, and functional implications of sphingolipids in inflammatory bowel disease (IBD), focusing on the different aspects of sphingolipid actions on inflammatory responses and on the potential of sphingolipid-targeted molecules as anti-IBD therapeutic agents. PMID:26880864

  3. Phase behavior of carbamyloxyphosphatidylcholine, a sphingolipid analogue.

    PubMed

    Curatolo, W; Bali, A; Gupta, C M

    1985-12-01

    The phase behavior of two series of phosphatidylcholines (PC) possessing carbamyloxy-linked fatty acids has been studied by differential scanning calorimetry. These non-natural phosphatidylcholines are of interest because they possess the capability of forming interlipid hydrogen bonds via the carbamyloxy function and as such can serve as a model for sphingolipids, the phase behavior of which is thought to be dominated by interlipid hydrogen bonding. Furthermore, carbamyloxyphosphatidylcholines form unusually stable liposomes, and thus have potential in drug delivery. Carbamyloxyphosphatidylcholines of the type di-(CnH2n+1NHCOO)-PC, where n = 13, 15, or 17, exhibit metastable polymorphism. Cooling from the liquid crystalline state results in formation of a metastable low-temperature polymorph I, which must transform into a stable low-temperature polymorph II before the liquid crystalline state can be reached again. Carbamyloxyphosphatidylcholines of the type 1-C16:0-2-(CnH2n+1NHCOO)-PC exhibit similar metastable polymorphism. This metastability is similar to that exhibited by certain cerebrosides and sphingomyelins and indicates that the sphingosine backbone is not a prerequisite for this type of metastability. Furthermore, the carbamyloxy group is reversed in orientation compared with the amide of sphingolipids (-NHCO- versus -CONH-), suggesting that the intermolecular hydrogen bonding potential, rather than some highly specific steric or conformational constraint, is responsible for the observed metastability of sphingolipids.

  4. Analysis of sphingolipid classes and their contents in meals.

    PubMed

    Yunoki, Keita; Ogawa, Takuya; Ono, Jisaburo; Miyashita, Rumiko; Aida, Kazuhiko; Oda, Yuji; Ohnishi, Masao

    2008-01-01

    Sphingolipids have attracted attention as physiologically functional lipids. We determined their class and content in Japanese meals that had been prepared by a nutritionist, mainly by using HPLC-ELSD. In all 12 meals tested, cerebroside and/or sphingomyelin were generally detected as the major sphingolipids. The total amounts of sphingolipids in typical high- and low-calorie meal samples over 2 days were 292 and 128 mg/day, and 81 and 45 mg/day, respectively.

  5. Phospholipid and sphingolipid metabolism in Leishmania

    PubMed Central

    Zhang, Kai; Beverley, Stephen M.

    2009-01-01

    In many eukaryotes, phospholipids (PLs) and sphingolipids (SLs) are abundant membrane components and reservoirs for important signaling molecules. In Leishmania, the composition, metabolism, and function of PLs and SLs differ significantly from those in mammalian cells. Although only a handful of enzymes have been experimentally characterized, available data suggest many steps of PL/SL metabolism are critical for Leishmania viability and/or virulence, and could be a source for new drug targets. Further studies of genes involved in the synthesis (de novo and salvage) and degradation of PLs and SLs will reveal their diverse effects on Leishmania pathogenesis. PMID:20026359

  6. A sphingolipid mechanism for behavioral extinction.

    PubMed

    Huston, Joseph P; Kornhuber, Johannes; Mühle, Christiane; Japtok, Lukasz; Komorowski, Mara; Mattern, Claudia; Reichel, Martin; Gulbins, Erich; Kleuser, Burkhard; Topic, Bianca; De Souza Silva, Maria A; Müller, Christian P

    2016-05-01

    Reward-dependent instrumental behavior must continuously be re-adjusted according to environmental conditions. Failure to adapt to changes in reward contingencies may incur psychiatric disorders like anxiety and depression. When an expected reward is omitted, behavior undergoes extinction. While extinction involves active re-learning, it is also accompanied by emotional behaviors indicative of frustration, anxiety, and despair (extinction-induced depression). Here, we report evidence for a sphingolipid mechanism in the extinction of behavior. Rapid extinction, indicating efficient re-learning, coincided with a decrease in the activity of the enzyme acid sphingomyelinase (ASM), which catalyzes turnover of sphingomyelin to ceramide, in the dorsal hippocampus of rats. The stronger the decline in ASM activity, the more rapid was the extinction. Sphingolipid-focused lipidomic analysis showed that this results in a decline of local ceramide species in the dorsal hippocampus. Ceramides shape the fluidity of lipid rafts in synaptic membranes and by that way can control neural plasticity. We also found that aging modifies activity of enzymes and ceramide levels in selective brain regions. Aging also changed how the chronic treatment with corticosterone (stress) or intranasal dopamine modified regional enzyme activity and ceramide levels, coinciding with rate of extinction. These data provide first evidence for a functional ASM-ceramide pathway in the brain involved in the extinction of learned behavior. This finding extends the known cellular mechanisms underlying behavioral plasticity to a new class of membrane-located molecules, the sphingolipids, and their regulatory enzymes, and may offer new treatment targets for extinction- and learning-related psychopathological conditions. Sphingolipids are common lipids in the brain which form lipid domains at pre- and postsynaptic membrane compartments. Here we show a decline in dorsal hippocampus ceramide species together with a

  7. Drug targeting of sphingolipid metabolism: sphingomyelinases and ceramidases

    PubMed Central

    Canals, Daniel; Perry, David M; Jenkins, Russell W; Hannun, Yusuf A

    2011-01-01

    Sphingolipids represent a class of diverse bioactive lipid molecules that are increasingly appreciated as key modulators of diverse physiologic and pathophysiologic processes that include cell growth, cell death, autophagy, angiogenesis, and stress and inflammatory responses. Sphingomyelinases and ceramidases are key enzymes of sphingolipid metabolism that regulate the formation and degradation of ceramide, one of the most intensely studied classes of sphingolipids. Improved understanding of these enzymes that control not only the levels of ceramide but also the complex interconversion of sphingolipid metabolites has provided the foundation for the functional analysis of the roles of sphingolipids. Our current understanding of the roles of various sphingolipids in the regulation of different cellular processes has come from loss-of-function/gain-of-function studies utilizing genetic deletion/downregulation/overexpression of enzymes of sphingolipid metabolism (e.g. knockout animals, RNA interference) and from the use of pharmacologic inhibitors of these same enzymes. While genetic approaches to evaluate the functional roles of sphingolipid enzymes have been instrumental in advancing the field, the use of pharmacologic inhibitors has been equally important in identifying new roles for sphingolipids in important cellular processes.The latter also promises the development of novel therapeutic targets with implications for cancer therapy, inflammation, diabetes, and neurodegeneration. In this review, we focus on the status and use of pharmacologic compounds that inhibit sphingomyelinases and ceramidases, and we will review the history, current uses and future directions for various small molecule inhibitors, and will highlight studies in which inhibitors of sphingolipid metabolizing enzymes have been used to effectively treat models of human disease. PMID:21615386

  8. Differential regulation of sphingolipid metabolism in plasma, hippocampus, and cerebral cortex of mice administered sphingolipid modulating agents.

    PubMed

    Giles, Corey; Takechi, Ryusuke; Mellett, Natalie A; Meikle, Peter J; Dhaliwal, Satvinder; Mamo, John C

    2017-05-01

    Accumulation of ceramide is implicated in mediating the cellular responses to stress and aberrant sphingolipid metabolism is frequently associated with metabolic and neurodegenerative diseases. It is often assumed that (i) peripheral disturbances in sphingolipid concentrations are reflective of processes occurring in the brain, or (ii) circulating sphingolipids directly influence cerebral sphingolipid abundance. In order to address these assumptions, this study explores, in a physiological system, the metabolic pathways regulating sphingolipid metabolism in the brain and plasma of mice. Male C57Bl/6 were maintained on a low fat (control diet) or saturated fat enriched (SFA) diet with, or without the provision of sphingolipid modulating agents. Following 6 months of feeding, the abundance of seven sphingolipid classes was assessed by LC-ESI-MS/MS in the hippocampus (HPF), cerebral cortex (CTX), and plasma. Long-term consumption of the SFA diet increased ceramide and dihydroceramide in the plasma. Inhibiting de novo synthesis ameliorated this effect, while inhibition of acidic sphingomyelinase, or the sphingosine-1-phosphate receptor agonist did not. SFA feeding did not influence sphingolipid levels in either the HPF or CTX. De novo synthesis inhibition reduced ceramide in the CTX, while treatment with a sphingosine-1-phosphate receptor agonist reduced ceramides in the HPF. Analysis of the individual ceramide species revealed the effects were chain-length dependent. Both positive and negative correlations were observed between plasma and HPF/CTX ceramide species. The findings in this study show that HPF and CTX sphingolipid concentration are influenced by distinct pathways, independent of peripheral sphingolipid concentration. © 2017 International Society for Neurochemistry.

  9. Sphingolipids are required for mammalian epidermal barrier function. Inhibition of sphingolipid synthesis delays barrier recovery after acute perturbation.

    PubMed Central

    Holleran, W M; Man, M Q; Gao, W N; Menon, G K; Elias, P M; Feingold, K R

    1991-01-01

    Stratum corneum lipids comprise an approximately equimolar mixture of sphingolipids, cholesterol, and free fatty acids, arranged as intercellular membrane bilayers that are presumed to mediate the epidermal permeability barrier. Prior studies have shown that alterations in epidermal barrier function lead to a rapid increase in cholesterol and fatty acid synthesis which parallels the early stages of the repair process. Despite an abundance of indirect evidence for their role in the barrier, the importance of sphingolipids has yet to be demonstrated directly. Whereas sphingolipid synthesis also increases during barrier repair, this response is delayed in comparison to cholesterol and fatty acid synthesis (Holleran, W.M., et al. 1991. J. Lipid Res. 32:1151-1158). To further delineate the role of sphingolipids in barrier homeostasis, we assessed the impact of inhibition of sphingolipid synthesis on epidermal barrier recovery. A single topical application of beta-chloro-L-alanine (beta-CA), an irreversible inhibitor of serine-palmitoyl transferase (SPT), applied to acetone-treated skin of hairless mice resulted in: (a) greater than 75% inhibition of SPT activity at 30 min (P less than 0.001); (b) a global decrease in sphingolipid synthesis between 1 and 3 h (P less than 0.02); (c) reduction of epidermal sphingolipid content at 18 h (P less than 0.01); (d) delayed reaccumulation of histochemical staining for sphingolipids in the stratum corneum; and (e) reduced numbers and contents of lamellar bodies in the stratum granulosum. Finally, despite its immediate, marked diminution of sphingolipid synthesis, beta-CA slowed barrier recovery only at late time points (greater than 6 h) after acetone treatment. This inhibition was overridden by coapplications of ceramides (the distal SPT product), indicating that the delay in repair was not due to non-specific toxicity. These studies demonstrate a distinctive role for epidermal sphingolipids in permeability barrier homeostasis

  10. Sphingolipid Metabolism, Oxidant Signaling, and Contractile Function of Skeletal Muscle

    PubMed Central

    Nikolova-Karakashian, Mariana N.

    2011-01-01

    Abstract Significance Sphingolipids are a class of bioactive lipids that regulate diverse cell functions. Ceramide, sphingosine, and sphingosine-1-phosphate accumulate in tissues such as liver, brain, and lung under conditions of cellular stress, including oxidative stress. The activity of some sphingolipid metabolizing enzymes, chiefly the sphingomyelinases, is stimulated during inflammation and in response to oxidative stress. Ceramide, the sphingomyelinase product, as well as the ceramide metabolite, sphingosine-1-phosphate, can induce the generation of more reactive oxygen species, propagating further inflammation. Recent Advances This review article summarizes information on sphingolipid biochemistry and signaling pertinent to skeletal muscle and describes the potential influence of sphingolipids on contractile function. Critical Issues It encompasses topics related to (1) the pathways for complex sphingolipid biosynthesis and degradation, emphasizing sphingolipid regulation in various muscle fiber types and subcellular compartments; (2) the emerging evidence that implicates ceramide, sphingosine, and sphingosine-1-phosphate as regulators of muscle oxidant activity, and (3) sphingolipid effects on contractile function and fatigue. Future Directions We propose that prolonged inflammatory conditions alter ceramide, sphingosine, and sphingosine-1-phosphate levels in skeletal muscle and that these changes promote the weakness, premature fatigue, and cachexia that plague individuals with heart failure, cancer, diabetes, and other chronic inflammatory diseases. Antioxid. Redox Signal. 15, 2501–2517. PMID:21453197

  11. Sphingolipid signalling in the cardiovascular system: good, bad or both?

    PubMed

    Alewijnse, Astrid E; Peters, Stephan L M

    2008-05-13

    Sphingolipids are biologically active lipids that play important roles in various cellular processes and the sphingomyelin metabolites ceramide, sphingosine and sphingosine-1-phosphate can act as signalling molecules in most cell types. With the recent development of the immunosuppressant drug FTY720 (Fingolimod) which after phosphorylation in vivo acts as a sphingosine-1-phosphate receptor agonist, research on the role of sphingolipids in the immune and other organ systems was triggered enormously. Since it was reported that FTY720 induced a modest, but significant transient decrease in heart rate in animals and humans, the question was raised which pharmacological properties of drugs targeting sphingolipid signalling will affect cardiovascular function in vivo. The answer to this question will most likely also indicate what type of drug could be used to treat cardiovascular disease. The latter is becoming increasingly important because of the increasing population carrying characteristics of the metabolic syndrome. This syndrome is, amongst others, characterized by obesity, hypertension, atherosclerosis and diabetes. As such, individuals with this syndrome are at increased risk of heart disease. Now numerous studies have investigated sphingolipid effects in the cardiovascular system, can we speculate whether certain sphingolipids under specific conditions are good, bad or maybe both? In this review we will give a brief overview of the pathophysiological role of sphingolipids in cardiovascular disease. In addition, we will try to answer how drugs that target sphingolipid signalling will potentially influence cardiovascular function and whether these drugs would be useful to treat cardiovascular disease.

  12. Sphingolipids and expression regulation of genes in cancer

    PubMed Central

    Patwardhan, Gauri A.; Liu, Yong-Yu

    2010-01-01

    Sphingolipids including glycosphingolipids have myriad effects on cell functions and affect cancer in aspects of tumorigenesis, metastasis and tumor response to treatments. Bioactive ones like ceramide, sphingosine 1-phosphate and globotriaosylceramide initiate and process cellular signaling to alter cell behaviors immediately responding to oncogenic stress or treatment challenges. Recent studies pinpoint that sphingolipid-mediated gene expression has long and profound impacts on cancer cells, and these play crucial roles in tumor progression and treatment outcome. More than ten sphingolipids and glycosphingolipids selectively mediate expressions of approximate fifty genes including c-myc, p21, c-fos, telomerase reverse transcriptase, caspase-9, Bcl-x, cyclooxygenase-2, matrix metalloproteinases, integrins, Oct-4, glucosylceramide synthase and multidrug-resistant gene 1. By diverse functions of these genes, sphingolipids enduringly affect cellular processes of mitosis, apoptosis, migration, stemness of cancer stem cells and cellular resistance to therapies. Mechanistic studies indicate that sphingolipids regulate particular gene expression by modulating phosphorylation and acetylation of proteins that serve as transcription factors (β-catenin, Sp1), repressor of transcription (histone H3), and regulators (SRp30a) in RNA splicing. Disclosing molecular mechanisms by which sphingolipids selectively regulate particular gene expression, instead of other relevant ones, requires understanding of the exact roles of individual lipid instead of a group, the signaling pathways that are implicated in and interaction with proteins or other lipids in details. These studies not only expand our knowledge of sphingolipids, but can also suggest novel targets for cancer treatments. PMID:20970453

  13. CFTR and sphingolipids mediate hypoxic pulmonary vasoconstriction

    PubMed Central

    Tabeling, Christoph; Yu, Hanpo; Wang, Liming; Ranke, Hannes; Goldenberg, Neil M.; Zabini, Diana; Noe, Elena; Krauszman, Adrienn; Gutbier, Birgitt; Yin, Jun; Schaefer, Michael; Arenz, Christoph; Hocke, Andreas C.; Suttorp, Norbert; Proia, Richard L.; Witzenrath, Martin; Kuebler, Wolfgang M.

    2015-01-01

    Hypoxic pulmonary vasoconstriction (HPV) optimizes pulmonary ventilation-perfusion matching in regional hypoxia, but promotes pulmonary hypertension in global hypoxia. Ventilation-perfusion mismatch is a major cause of hypoxemia in cystic fibrosis. We hypothesized that cystic fibrosis transmembrane conductance regulator (CFTR) may be critical in HPV, potentially by modulating the response to sphingolipids as mediators of HPV. HPV and ventilation-perfusion mismatch were analyzed in isolated mouse lungs or in vivo. Ca2+ mobilization and transient receptor potential canonical 6 (TRPC6) translocation were studied in human pulmonary (PASMCs) or coronary (CASMCs) artery smooth muscle cells. CFTR inhibition or deficiency diminished HPV and aggravated ventilation-perfusion mismatch. In PASMCs, hypoxia caused CFTR to interact with TRPC6, whereas CFTR inhibition attenuated hypoxia-induced TRPC6 translocation to caveolae and Ca2+ mobilization. Ca2+ mobilization by sphingosine-1-phosphate (S1P) was also attenuated by CFTR inhibition in PASMCs, but amplified in CASMCs. Inhibition of neutral sphingomyelinase (nSMase) blocked HPV, whereas exogenous nSMase caused TRPC6 translocation and vasoconstriction that were blocked by CFTR inhibition. nSMase- and hypoxia-induced vasoconstriction, yet not TRPC6 translocation, were blocked by inhibition or deficiency of sphingosine kinase 1 (SphK1) or antagonism of S1P receptors 2 and 4 (S1P2/4). S1P and nSMase had synergistic effects on pulmonary vasoconstriction that involved TRPC6, phospholipase C, and rho kinase. Our findings demonstrate a central role of CFTR and sphingolipids in HPV. Upon hypoxia, nSMase triggers TRPC6 translocation, which requires its interaction with CFTR. Concomitant SphK1-dependent formation of S1P and activation of S1P2/4 result in phospholipase C-mediated TRPC6 and rho kinase activation, which conjointly trigger vasoconstriction. PMID:25829545

  14. CFTR and sphingolipids mediate hypoxic pulmonary vasoconstriction.

    PubMed

    Tabeling, Christoph; Yu, Hanpo; Wang, Liming; Ranke, Hannes; Goldenberg, Neil M; Zabini, Diana; Noe, Elena; Krauszman, Adrienn; Gutbier, Birgitt; Yin, Jun; Schaefer, Michael; Arenz, Christoph; Hocke, Andreas C; Suttorp, Norbert; Proia, Richard L; Witzenrath, Martin; Kuebler, Wolfgang M

    2015-03-31

    Hypoxic pulmonary vasoconstriction (HPV) optimizes pulmonary ventilation-perfusion matching in regional hypoxia, but promotes pulmonary hypertension in global hypoxia. Ventilation-perfusion mismatch is a major cause of hypoxemia in cystic fibrosis. We hypothesized that cystic fibrosis transmembrane conductance regulator (CFTR) may be critical in HPV, potentially by modulating the response to sphingolipids as mediators of HPV. HPV and ventilation-perfusion mismatch were analyzed in isolated mouse lungs or in vivo. Ca(2+) mobilization and transient receptor potential canonical 6 (TRPC6) translocation were studied in human pulmonary (PASMCs) or coronary (CASMCs) artery smooth muscle cells. CFTR inhibition or deficiency diminished HPV and aggravated ventilation-perfusion mismatch. In PASMCs, hypoxia caused CFTR to interact with TRPC6, whereas CFTR inhibition attenuated hypoxia-induced TRPC6 translocation to caveolae and Ca(2+) mobilization. Ca(2+) mobilization by sphingosine-1-phosphate (S1P) was also attenuated by CFTR inhibition in PASMCs, but amplified in CASMCs. Inhibition of neutral sphingomyelinase (nSMase) blocked HPV, whereas exogenous nSMase caused TRPC6 translocation and vasoconstriction that were blocked by CFTR inhibition. nSMase- and hypoxia-induced vasoconstriction, yet not TRPC6 translocation, were blocked by inhibition or deficiency of sphingosine kinase 1 (SphK1) or antagonism of S1P receptors 2 and 4 (S1P2/4). S1P and nSMase had synergistic effects on pulmonary vasoconstriction that involved TRPC6, phospholipase C, and rho kinase. Our findings demonstrate a central role of CFTR and sphingolipids in HPV. Upon hypoxia, nSMase triggers TRPC6 translocation, which requires its interaction with CFTR. Concomitant SphK1-dependent formation of S1P and activation of S1P2/4 result in phospholipase C-mediated TRPC6 and rho kinase activation, which conjointly trigger vasoconstriction.

  15. Viral trans-dominant manipulation of algal sphingolipids.

    PubMed

    Michaelson, Louise V; Dunn, Teresa M; Napier, Johnathan A

    2010-12-01

    Emiliania huxleyi is the host for the coccolithovirus (EhV), which is responsible for the demise of large oceanic blooms formed by this alga. The EhV-86 virus genome sequence has identified several genes apparently involved in sphingolipid metabolism. Recently, an unusual glucosylceramide from E. huxleyi infected with EhV-86 was isolated, implicating sphingolipids in the lysis of this alga. However, the EhV-86-encoded genes contain only a subset of the activities required to generate the novel sphingolipid, implying that its synthesis is the result of coordinated interactions between algal- and viral-encoded biosynthetic enzymes. Here, we discuss the likely role for EhV-86 open reading frames (ORFs) in the synthesis of novel sphingolipids and also consider the concept of the trans-dominant manipulation of lipid metabolism.

  16. Tamoxifen regulation of sphingolipid metabolism—therapeutic implications

    PubMed Central

    Morad, Samy A F; Cabot, Myles C

    2015-01-01

    Tamoxifen, a triphenylethylene antiestrogen and one of the first-line endocrine therapies used to treat estrogen receptor-positive breast cancer, has a number of interesting, off-target effects, and among these is the inhibition of sphingolipid metabolism. More specifically, tamoxifen inhibits ceramide glycosylation, and enzymatic step that can adventitiously support the influential tumor-suppressor properties of ceramide, the aliphatic backbone of sphingolipids. Additionally, tamoxifen and metabolites N-desmethyltamoxifen and 4-hydroxytamoxifen, have been shown to inhibit ceramide hydrolysis by the enzyme acid ceramidase. This particular intervention slows ceramide destruction and thereby depresses formation of sphingosine 1-phosphate, a mitogenic sphingolipid with cancer growth-promoting properties. As ceramide-centric therapies are becoming appealing clinical interventions in the treatment of cancer, agents like tamoxifen that can retard the generation of mitogenic sphingolipids and buffer ceramide clearance via inhibition of glycosylation, take on new importance. In this review, we present an abridged, lay introduction to sphingolipid metabolism, briefly chronicle tamoxifen’s history in the clinic, examine studies that demonstrate the impact of triphenylethylenes on sphingolipid metabolism in cancer cells, and canvass works relevant to the use of tamoxifen as adjuvant to drive ceramide-centric therapies in cancer treatment. The objective is to inform the readership of what could be a novel, off-label indication of tamoxifen and structurally-related triphenylethylenes, an indication divorced from estrogen receptor status and one with application in drug resistance. PMID:25964209

  17. Involvement of sphingolipids in ethanol neurotoxicity in the developing brain.

    PubMed

    Saito, Mariko; Saito, Mitsuo

    2013-04-26

    Ethanol-induced neuronal death during a sensitive period of brain development is considered one of the significant causes of fetal alcohol spectrum disorders (FASD). In rodent models, ethanol triggers robust apoptotic neurodegeneration during a period of active synaptogenesis that occurs around the first two postnatal weeks, equivalent to the third trimester in human fetuses. The ethanol-induced apoptosis is mitochondria-dependent, involving Bax and caspase-3 activation. Such apoptotic pathways are often mediated by sphingolipids, a class of bioactive lipids ubiquitously present in eukaryotic cellular membranes. While the central role of lipids in ethanol liver toxicity is well recognized, the involvement of sphingolipids in ethanol neurotoxicity is less explored despite mounting evidence of their importance in neuronal apoptosis. Nevertheless, recent studies indicate that ethanol-induced neuronal apoptosis in animal models of FASD is mediated or regulated by cellular sphingolipids, including via the pro-apoptotic action of ceramide and through the neuroprotective action of GM1 ganglioside. Such sphingolipid involvement in ethanol neurotoxicity in the developing brain may provide unique targets for therapeutic applications against FASD. Here we summarize findings describing the involvement of sphingolipids in ethanol-induced apoptosis and discuss the possibility that the combined action of various sphingolipids in mitochondria may control neuronal cell fate.

  18. Sphingolipid metabolism and its role in the skeletal tissues.

    PubMed

    Khavandgar, Zohreh; Murshed, Monzur

    2015-03-01

    The regulators affecting skeletal tissue formation and its maintenance include a wide array of molecules with very diverse functions. More recently, sphingolipids have been added to this growing list of regulatory molecules in the skeletal tissues. Sphingolipids are integral parts of various lipid membranes present in the cells and organelles. For a long time, these macromolecules were considered as inert structural elements. This view, however, has radically changed in recent years as sphingolipids are now recognized as important second messengers for signal-transduction pathways that affect cell growth, differentiation, stress responses and programmed death. In the current review, we discuss the available data showing the roles of various sphingolipids in three different skeletal cell types-chondrocytes in cartilage and osteoblasts and osteoclasts in bone. We provide an overview of the biology of sphingomyelin phosphodiesterase 3 (SMPD3), an important regulator of sphingolipid metabolism in the skeleton. SMPD3 is localized in the plasma membrane and has been shown to cleave sphingomyelin to generate ceramide, a bioactive lipid second messenger, and phosphocholine, an essential nutrient. SMPD3 deficiency in mice impairs the mineralization in both cartilage and bone extracellular matrices leading to severe skeletal deformities. A detailed understanding of SMPD3 function may provide a novel insight on the role of sphingolipids in the skeletal tissues.

  19. Tamoxifen regulation of sphingolipid metabolism--Therapeutic implications.

    PubMed

    Morad, Samy A F; Cabot, Myles C

    2015-09-01

    Tamoxifen, a triphenylethylene antiestrogen and one of the first-line endocrine therapies used to treat estrogen receptor-positive breast cancer, has a number of interesting, off-target effects, and among these is the inhibition of sphingolipid metabolism. More specifically, tamoxifen inhibits ceramide glycosylation, and enzymatic step that can adventitiously support the influential tumor-suppressor properties of ceramide, the aliphatic backbone of sphingolipids. Additionally, tamoxifen and metabolites N-desmethyltamoxifen and 4-hydroxytamoxifen, have been shown to inhibit ceramide hydrolysis by the enzyme acid ceramidase. This particular intervention slows ceramide destruction and thereby depresses formation of sphingosine 1-phosphate, a mitogenic sphingolipid with cancer growth-promoting properties. As ceramide-centric therapies are becoming appealing clinical interventions in the treatment of cancer, agents like tamoxifen that can retard the generation of mitogenic sphingolipids and buffer ceramide clearance via inhibition of glycosylation, take on new importance. In this review, we present an abridged, lay introduction to sphingolipid metabolism, briefly chronicle tamoxifen's history in the clinic, examine studies that demonstrate the impact of triphenylethylenes on sphingolipid metabolism in cancer cells, and canvass works relevant to the use of tamoxifen as adjuvant to drive ceramide-centric therapies in cancer treatment. The objective is to inform the readership of what could be a novel, off-label indication of tamoxifen and structurally-related triphenylethylenes, an indication divorced from estrogen receptor status and one with application in drug resistance.

  20. Involvement of Sphingolipids in Ethanol Neurotoxicity in the Developing Brain

    PubMed Central

    Saito, Mariko; Saito, Mitsuo

    2013-01-01

    Ethanol-induced neuronal death during a sensitive period of brain development is considered one of the significant causes of fetal alcohol spectrum disorders (FASD). In rodent models, ethanol triggers robust apoptotic neurodegeneration during a period of active synaptogenesis that occurs around the first two postnatal weeks, equivalent to the third trimester in human fetuses. The ethanol-induced apoptosis is mitochondria-dependent, involving Bax and caspase-3 activation. Such apoptotic pathways are often mediated by sphingolipids, a class of bioactive lipids ubiquitously present in eukaryotic cellular membranes. While the central role of lipids in ethanol liver toxicity is well recognized, the involvement of sphingolipids in ethanol neurotoxicity is less explored despite mounting evidence of their importance in neuronal apoptosis. Nevertheless, recent studies indicate that ethanol-induced neuronal apoptosis in animal models of FASD is mediated or regulated by cellular sphingolipids, including via the pro-apoptotic action of ceramide and through the neuroprotective action of GM1 ganglioside. Such sphingolipid involvement in ethanol neurotoxicity in the developing brain may provide unique targets for therapeutic applications against FASD. Here we summarize findings describing the involvement of sphingolipids in ethanol-induced apoptosis and discuss the possibility that the combined action of various sphingolipids in mitochondria may control neuronal cell fate. PMID:24961420

  1. Role of Sphingolipids in the Pathobiology of Lung Inflammation

    PubMed Central

    Ghidoni, Riccardo; Caretti, Anna; Signorelli, Paola

    2015-01-01

    Sphingolipid bioactivities in the respiratory airways and the roles of the proteins that handle them have been extensively investigated. Gas or inhaled particles or microorganisms come into contact with mucus components, epithelial cells, blood barrier, and immune surveillance within the airways. Lung structure and functionality rely on a complex interplay of polar and hydrophobic structures forming the surfactant layer and governing external-internal exchanges, such as glycerol-phospholipids sphingolipids and proteins. Sphingolipids act as important signaling mediators involved in the control of cell survival and stress response, as well as secreted molecules endowed with inflammation-regulatory activities. Most successful respiratory infection and injuries evolve in the alveolar compartment, the critical lung functional unit involved in gas exchange. Sphingolipid altered metabolism in this compartment is closely related to inflammatory reaction and ceramide increase, in particular, favors the switch to pathological hyperinflammation. This short review explores a few mechanisms underlying sphingolipid involvement in the healthy lung (surfactant production and endothelial barrier maintenance) and in a selection of lung pathologies in which the impact of sphingolipid synthesis and metabolism is most apparent, such as acute lung injury, or chronic pathologies such as cystic fibrosis and chronic obstructive pulmonary disease. PMID:26770018

  2. Ceramide synthases at the centre of sphingolipid metabolism and biology.

    PubMed

    Mullen, Thomas D; Hannun, Yusuf A; Obeid, Lina M

    2012-02-01

    Sphingolipid metabolism in metazoan cells consists of a complex interconnected web of numerous enzymes, metabolites and modes of regulation. At the centre of sphingolipid metabolism reside CerSs (ceramide synthases), a group of enzymes that catalyse the formation of ceramides from sphingoid base and acyl-CoA substrates. From a metabolic perspective, these enzymes occupy a unique niche in that they simultaneously regulate de novo sphingolipid synthesis and the recycling of free sphingosine produced from the degradation of pre-formed sphingolipids (salvage pathway). Six mammalian CerSs (CerS1-CerS6) have been identified. Unique characteristics have been described for each of these enzymes, but perhaps the most notable is the ability of individual CerS isoforms to produce ceramides with characteristic acyl-chain distributions. Through this control of acyl-chain length and perhaps in a compartment-specific manner, CerSs appear to regulate multiple aspects of sphingolipid-mediated cell and organismal biology. In the present review, we discuss the function of CerSs as critical regulators of sphingolipid metabolism, highlight their unique characteristics and explore the emerging roles of CerSs in regulating programmed cell death, cancer and many other aspects of biology.

  3. Ceramide synthases at the centre of sphingolipid metabolism and biology

    PubMed Central

    Mullen, Thomas D.; Hannun, Yusuf A.; Obeid, Lina M.

    2013-01-01

    Sphingolipid metabolism in metazoan cells consists of a complex interconnected web of numerous enzymes, metabolites and modes of regulation. At the centre of sphingolipid metabolism reside CerSs (ceramide synthases), a group of enzymes that catalyse the formation of ceramides from sphingoid base and acyl-CoA substrates. From a metabolic perspective, these enzymes occupy a unique niche in that they simultaneously regulate de novo sphingolipid synthesis and the recycling of free sphingosine produced from the degradation of pre-formed sphingolipids (salvage pathway). Six mammalian CerSs (CerS1–CerS6) have been identified. Unique characteristics have been described for each of these enzymes, but perhaps the most notable is the ability of individual CerS isoforms to produce ceramides with characteristic acyl-chain distributions. Through this control of acyl-chain length and perhaps in a compartment-specific manner, CerSs appear to regulate multiple aspects of sphingolipid-mediated cell and organismal biology. In the present review, we discuss the function of CerSs as critical regulators of sphingolipid metabolism, highlight their unique characteristics and explore the emerging roles of CerSs in regulating programmed cell death, cancer and many other aspects of biology. PMID:22248339

  4. Lipoxygenase inhibitory sphingolipids from Launaea nudicaulis.

    PubMed

    Riaz, Naheed; Parveen, Shehla; Saleem, Muhammad; Ali, Muhammad Shaiq; Malik, Abdul; Ashraf, Muhammad; Afzal, Iftikhar; Jabbar, Abdul

    2012-01-01

    Four new sphingolipids: nudicaulin A [(2S,3S,4R,14E)-2-{[octadecanoyl]amino}tetraeicos-14-ene-1,3,4-triol; 1], nudicaulin B [(2S,3S,4R,14E)-2-{[(2R)-2-hydroxyoctadecanoyl]amino}tetraeicos-14-ene-1,3,4-triol; 2], nudicaulin C [(2S,3S,4R,14E)-2-{[(2R)-2-hydroxyoctadecanoyl]amino}tetraeicos-14-ene-1,3,4-triol-1-O-β-D-glucopyranoside; 3], and nudicaulin D [(2S,3S,4R)-2-{[(2R,3S,12E)-2,3-dihydroxyeicos-12-enoyl]amino}octadecane-1,3,4-triol; 4] together with 1-hexatriacontanol, β-sitosterol, octadecyl 4-hydroxycinnamate, elaidic acid, cholesta-5,22-diene-3,7-diol, oleanolic acid, apigenin, and β-sitosterol 3-O-β-D-glucopyranoside were isolated from the methanolic extract of the whole plant of Launaea nudicaulis. Their structures were elucidated using ¹H and ¹³C NMR spectra and 2D NMR analyses (HMQC, HMBC, and COSY) in combination with mass spectrometry (EI-MS, HR-EI-MS, FAB-MS, and HR-FAB-MS) experiments and comparison with literature data of related compounds. Compounds 1-4 displayed moderate inhibitory potential against enzyme lipoxygenase in concentration-dependent manner with IC₅₀ value ranges 103-193 μM.

  5. Sphingolipids: the nexus between Gaucher disease and insulin resistance.

    PubMed

    Fuller, Maria

    2010-10-11

    Sphingolipids constitute a diverse array of lipids in which fatty acids are linked through amide bonds to a long-chain base, and, structurally, they form the building blocks of eukaryotic membranes. Ceramide is the simplest and serves as a precursor for the synthesis of the three main types of complex sphingolipids; sphingomyelins, glycosphingolipids and gangliosides. Sphingolipids are no longer considered mere structural spectators, but bioactive molecules with functions beyond providing a mechanically stable and chemically resistant barrier to a diverse array of cellular processes. Although sphingolipids form a somewhat minor component of the total cellular lipid pool, their accumulation in certain cells forms the basis of many diseases. Human diseases caused by alterations in the metabolism of sphingolipids are conventionally inborn errors of degradation, the most common being Gaucher disease, in which the catabolism of glucosylceramide is defective and accumulates. Insulin resistance has been reported in patients with Gaucher disease and this article presents evidence that this is due to perturbations in the metabolism of sphingolipids. Ceramide and the more complex sphingolipids, the gangliosides, are constituents of specialised membrane microdomains termed lipid rafts. Lipid rafts play a role in facilitating and regulating lipid and protein interactions in cells, and their unique lipid composition enables them to carry out this role. The lipid composition of rafts is altered in cell models of Gaucher disease which may be responsible for impaired lipid and protein sorting observed in this disorder, and consequently pathology. Lipid rafts are also necessary for correct insulin signalling, and a perturbed lipid raft composition may impair insulin signalling. Unravelling common nodes of interaction between insulin resistance and Gaucher disease may lead to a better understanding of the biochemical mechanisms behind pathology.

  6. The sphingolipid biosynthetic enzyme Sphingolipid delta8 desaturase is important for chilling resistance of tomato

    PubMed Central

    Zhou, Ying; Zeng, Lanting; Fu, Xiumin; Mei, Xin; Cheng, Sihua; Liao, Yinyin; Deng, Rufang; Xu, Xinlan; Jiang, Yueming; Duan, Xuewu; Baldermann, Susanne; Yang, Ziyin

    2016-01-01

    The physiological functions of sphingolipids in animals have been intensively studied, while less attention has been paid to their roles in plants. Here, we reveal the involvement of sphingolipid delta8 desaturase (SlSLD) in the chilling resistance of tomato (Solanum lycopersicum cv. Micro-Tom). We used the virus-induced gene silencing (VIGS) approach to knock-down SlSLD expression in tomato leaves, and then evaluated chilling resistance. Changes in leaf cell structure under a chilling treatment were observed by transmission electron microscopy. In control plants, SlSLD was highly expressed in the fruit and leaves in response to a chilling treatment. The degree of chilling damage was greater in SlSLD-silenced plants than in control plants, indicating that SlSLD knock-down significantly reduced the chilling resistance of tomato. Compared with control plants, SlSLD-silenced plants showed higher relative electrolytic leakage and malondialdehyde content, and lower superoxide dismutase and peroxidase activities after a chilling treatment. Chilling severely damaged the chloroplasts in SlSLD-silenced plants, resulting in the disruption of chloroplast membranes, swelling of thylakoids, and reduced granal stacking. Together, these results show that SlSLD is crucial for chilling resistance in tomato. PMID:27929095

  7. Disruption of Sphingolipid Biosynthesis Blocks Phagocytosis of Candida albicans

    PubMed Central

    Schmidt, Florian I.; Freinkman, Elizaveta; Dougan, Stephanie; Dougan, Michael; Esteban, Alexandre; Maruyama, Takeshi; Strijbis, Karin; Ploegh, Hidde L.

    2015-01-01

    The ability of phagocytes to clear pathogens is an essential attribute of the innate immune response. The role of signaling lipid molecules such as phosphoinositides is well established, but the role of membrane sphingolipids in phagocytosis is largely unknown. Using a genetic approach and small molecule inhibitors, we show that phagocytosis of Candida albicans requires an intact sphingolipid biosynthetic pathway. Blockade of serine-palmitoyltransferase (SPT) and ceramide synthase-enzymes involved in sphingolipid biosynthesis- by myriocin and fumonisin B1, respectively, impaired phagocytosis by phagocytes. We used CRISPR/Cas9-mediated genome editing to generate Sptlc2-deficient DC2.4 dendritic cells, which lack serine palmitoyl transferase activity. Sptlc2-/- DC2.4 cells exhibited a stark defect in phagocytosis, were unable to bind fungal particles and failed to form a normal phagocytic cup to engulf C. albicans. Supplementing the growth media with GM1, the major ganglioside present at the cell surface, restored phagocytic activity of Sptlc2-/- DC2.4 cells. While overall membrane trafficking and endocytic pathways remained functional, Sptlc2-/- DC2.4 cells express reduced levels of the pattern recognition receptors Dectin-1 and TLR2 at the cell surface. Consistent with the in vitro data, compromised sphingolipid biosynthesis in mice sensitizes the animal to C. albicans infection. Sphingolipid biosynthesis is therefore critical for phagocytosis and in vivo clearance of C. albicans. PMID:26431038

  8. Membrane engineering of S. cerevisiae targeting sphingolipid metabolism

    PubMed Central

    Lindahl, Lina; Santos, Aline X. S.; Olsson, Helén; Olsson, Lisbeth; Bettiga, Maurizio

    2017-01-01

    The sustainable production of fuels and chemicals using microbial cell factories is now well established. However, many microbial production processes are still limited in scale due to inhibition from compounds that are present in the feedstock or are produced during fermentation. Some of these inhibitors interfere with cellular membranes and change the physicochemical properties of the membranes. Another group of molecules is dependent on their permeation rate through the membrane for their inhibition. We have investigated the use of membrane engineering to counteract the negative effects of inhibitors on the microorganism with focus on modulating the abundance of complex sphingolipids in the cell membrane of Saccharomyces cerevisiae. Overexpression of ELO3, involved in fatty acid elongation, and AUR1, which catalyses the formation of complex sphingolipids, had no effect on the membrane lipid profile or on cellular physiology. Deletion of the genes ORM1 and ORM2, encoding negative regulators of sphingolipid biosynthesis, decreased cell viability and considerably reduced phosphatidylinositol and complex sphingolipids. Additionally, combining ELO3 and AUR1 overexpression with orm1/2Δ improved cell viability and increased fatty acyl chain length compared with only orm1/2Δ. These findings can be used to further study the sphingolipid metabolism, as well as giving guidance in membrane engineering. PMID:28145511

  9. Disruption of Sphingolipid Biosynthesis Blocks Phagocytosis of Candida albicans.

    PubMed

    Tafesse, Fikadu G; Rashidfarrokhi, Ali; Schmidt, Florian I; Freinkman, Elizaveta; Dougan, Stephanie; Dougan, Michael; Esteban, Alexandre; Maruyama, Takeshi; Strijbis, Karin; Ploegh, Hidde L

    2015-10-01

    The ability of phagocytes to clear pathogens is an essential attribute of the innate immune response. The role of signaling lipid molecules such as phosphoinositides is well established, but the role of membrane sphingolipids in phagocytosis is largely unknown. Using a genetic approach and small molecule inhibitors, we show that phagocytosis of Candida albicans requires an intact sphingolipid biosynthetic pathway. Blockade of serine-palmitoyltransferase (SPT) and ceramide synthase-enzymes involved in sphingolipid biosynthesis- by myriocin and fumonisin B1, respectively, impaired phagocytosis by phagocytes. We used CRISPR/Cas9-mediated genome editing to generate Sptlc2-deficient DC2.4 dendritic cells, which lack serine palmitoyl transferase activity. Sptlc2-/- DC2.4 cells exhibited a stark defect in phagocytosis, were unable to bind fungal particles and failed to form a normal phagocytic cup to engulf C. albicans. Supplementing the growth media with GM1, the major ganglioside present at the cell surface, restored phagocytic activity of Sptlc2-/- DC2.4 cells. While overall membrane trafficking and endocytic pathways remained functional, Sptlc2-/- DC2.4 cells express reduced levels of the pattern recognition receptors Dectin-1 and TLR2 at the cell surface. Consistent with the in vitro data, compromised sphingolipid biosynthesis in mice sensitizes the animal to C. albicans infection. Sphingolipid biosynthesis is therefore critical for phagocytosis and in vivo clearance of C. albicans.

  10. Sphingolipid metabolism regulates development and lifespan in Caenorhabditis elegans

    PubMed Central

    Cutler, Roy G.; Thompson, Kenneth W.; Camandola, Simonetta; Mack, Kendra T.; Mattson, Mark P.

    2015-01-01

    Sphingolipids are a highly conserved lipid component of cell membranes involved in the formation of lipid raft domains that house many of the receptors and cell-to-cell signaling factors involved in regulating cell division, maturation, and terminal differentiation. By measuring and manipulating sphingolipid metabolism using pharmacological and genetic tools in Caenorhabditis elegans, we provide evidence that the synthesis and remodeling of specific ceramides (e.g., dC18:1–C24:1), gangliosides (e.g., GM1–C24:1), and sphingomyelins (e.g., dC18:1–C18:1) influence development rate and lifespan. We found that the levels of fatty acid chain desaturation and elongation in many sphingolipid species increased during development and aging, with no such changes in developmentally-arrested dauer larvae or normal adults after food withdrawal (an anti-aging intervention). Pharmacological inhibitors and small interfering RNAs directed against serine palmitoyl transferase and glucosylceramide synthase acted to slow development rate, extend the reproductive period, and increase lifespan. In contrast, worms fed an egg yolk diet rich in sphingolipids exhibited accelerated development and reduced lifespan. Our findings demonstrate that sphingolipid accumulation and remodeling are critical events that determine development rate and lifespan in the nematode model, with both development rate and aging being accelerated by the synthesis of sphingomyelin, and its metabolism to ceramides and gangliosides. PMID:25437839

  11. Membrane engineering of S. cerevisiae targeting sphingolipid metabolism.

    PubMed

    Lindahl, Lina; Santos, Aline X S; Olsson, Helén; Olsson, Lisbeth; Bettiga, Maurizio

    2017-02-01

    The sustainable production of fuels and chemicals using microbial cell factories is now well established. However, many microbial production processes are still limited in scale due to inhibition from compounds that are present in the feedstock or are produced during fermentation. Some of these inhibitors interfere with cellular membranes and change the physicochemical properties of the membranes. Another group of molecules is dependent on their permeation rate through the membrane for their inhibition. We have investigated the use of membrane engineering to counteract the negative effects of inhibitors on the microorganism with focus on modulating the abundance of complex sphingolipids in the cell membrane of Saccharomyces cerevisiae. Overexpression of ELO3, involved in fatty acid elongation, and AUR1, which catalyses the formation of complex sphingolipids, had no effect on the membrane lipid profile or on cellular physiology. Deletion of the genes ORM1 and ORM2, encoding negative regulators of sphingolipid biosynthesis, decreased cell viability and considerably reduced phosphatidylinositol and complex sphingolipids. Additionally, combining ELO3 and AUR1 overexpression with orm1/2Δ improved cell viability and increased fatty acyl chain length compared with only orm1/2Δ. These findings can be used to further study the sphingolipid metabolism, as well as giving guidance in membrane engineering.

  12. Sphingolipids as Regulators of the Phagocytic Response to Fungal Infections

    PubMed Central

    Bryan, Arielle M.; Del Poeta, Maurizio; Luberto, Chiara

    2015-01-01

    Fungal infections pose a significant risk for the increasing population of individuals who are immunocompromised. Phagocytes play an important role in immune defense against fungal pathogens, but the interactions between host and fungi are still not well understood. Sphingolipids have been shown to play an important role in many cell functions, including the function of phagocytes. In this review, we discuss major findings that relate to the importance of sphingolipids in macrophage and neutrophil function and the role of macrophages and neutrophils in the most common types of fungal infections, as well as studies that have linked these three concepts to show the importance of sphingolipid signaling in immune response to fungal infections. PMID:26688618

  13. Sterols and sphingolipids: Dynamic duo or partners in crime?

    PubMed Central

    Gulati, Sonia; Liu, Ying; Munkacsi, Andrew B.; Wilcox, Lisa; Sturley, Stephen L.

    2010-01-01

    One manner in which eukaryotic cells respond to their environments is by optimizing the composition and proportions of sterols and sphingolipids in membranes. The physical association of the planar ring of sterols with the acyl chains of phospholipids, particularly sphingolipids, produces membrane micro-heterogeneity that is exploited to coordinate several crucial pathways. We hypothesize that these lipid molecules play an integrated role in human disease; when one of the partners is mis-regulated, pathology frequently ensues. Sterols and sphingolipid levels are not coordinated by the action of a single master regulator, however the cross talk between their metabolic pathways is considerable. We describe our perspectives on the key components of synthesis, catabolism and transport of these lipid partners with an emphasis on evolutionarily conserved reactions that produce disease states when defective. PMID:20362613

  14. Tumor PDT-associated immune response: relevance of sphingolipids

    NASA Astrophysics Data System (ADS)

    Korbelik, Mladen; Merchant, Soroush; Separovic, Duska M.

    2010-02-01

    Sphingolipids have become recognized as essential effector molecules in signal transduction with involvement in various aspects of cell function and death, immune response and cancer treatment response. Major representatives of sphingolipids family, ceramide, sphingosine and sphingosine-1-phosphate (S1P), have attracted interest in their relevance to tumor response to photodynamic therapy (PDT) because of their roles as enhancers of apoptosis, mediators of cell growth and vasculogenesis, and regulators of immune response. Our recent in vivo studies with mouse tumor models have confirmed that PDT treatment has a pronounced impact on sphingolipid profile in the targeted tumor and that significant advances in therapeutic gain with PDT can be attained by combining this modality with adjuvant treatment with ceramide analog LCL29.

  15. Sphingolipids as targets for treatment of fungal infections

    PubMed Central

    Rollin-Pinheiro, Rodrigo; Singh, Ashutosh; Barreto-Bergter, Eliana; Del Poeta, Maurizio

    2016-01-01

    Invasive fungal infections have significantly increased in the last few decades. Three classes of drugs are commonly used to treat these infections: polyenes, azoles and echinocandins. Unfortunately each of these drugs has drawbacks; polyenes are toxic, resistance against azoles is emerging and echinocandins have narrow spectrum of activity. Thus, the development of new antifungals is urgently needed. In this context, fungal sphingolipids have emerged as a potential target for new antifungals, because their biosynthesis in fungi is structurally different than in mammals. Besides, some fungal sphingolipids play an important role in the regulation of virulence in a variety of fungi. This review aims to highlight the diverse strategies that could be used to block the synthesis or/and function of fungal sphingolipids. PMID:27502288

  16. Genetic Determinants of Circulating Sphingolipid Concentrations in European Populations

    PubMed Central

    Pramstaller, Peter P.; Rudan, Igor; Franklin, Christopher S.; Liebisch, Gerhard; Erdmann, Jeanette; Jonasson, Inger; Zorkoltseva, Irina V.; Pattaro, Cristian; Hayward, Caroline; Isaacs, Aaron; Hengstenberg, Christian; Campbell, Susan; Gnewuch, Carsten; Janssens, A. CecileJ.W.; Kirichenko, Anatoly V.; König, Inke R.; Marroni, Fabio; Polasek, Ozren; Demirkan, Ayse; Kolcic, Ivana; Schwienbacher, Christine; Igl, Wilmar; Biloglav, Zrinka; Witteman, Jacqueline C. M.; Pichler, Irene; Zaboli, Ghazal; Axenovich, Tatiana I.; Peters, Annette; Schreiber, Stefan; Wichmann, H.-Erich; Schunkert, Heribert; Hastie, Nick; Oostra, Ben A.; Wild, Sarah H.; Meitinger, Thomas; Gyllensten, Ulf; van Duijn, Cornelia M.; Wilson, James F.; Wright, Alan; Schmitz, Gerd; Campbell, Harry

    2009-01-01

    Sphingolipids have essential roles as structural components of cell membranes and in cell signalling, and disruption of their metabolism causes several diseases, with diverse neurological, psychiatric, and metabolic consequences. Increasingly, variants within a few of the genes that encode enzymes involved in sphingolipid metabolism are being associated with complex disease phenotypes. Direct experimental evidence supports a role of specific sphingolipid species in several common complex chronic disease processes including atherosclerotic plaque formation, myocardial infarction (MI), cardiomyopathy, pancreatic β-cell failure, insulin resistance, and type 2 diabetes mellitus. Therefore, sphingolipids represent novel and important intermediate phenotypes for genetic analysis, yet little is known about the major genetic variants that influence their circulating levels in the general population. We performed a genome-wide association study (GWAS) between 318,237 single-nucleotide polymorphisms (SNPs) and levels of circulating sphingomyelin (SM), dihydrosphingomyelin (Dih-SM), ceramide (Cer), and glucosylceramide (GluCer) single lipid species (33 traits); and 43 matched metabolite ratios measured in 4,400 subjects from five diverse European populations. Associated variants (32) in five genomic regions were identified with genome-wide significant corrected p-values ranging down to 9.08×10−66. The strongest associations were observed in or near 7 genes functionally involved in ceramide biosynthesis and trafficking: SPTLC3, LASS4, SGPP1, ATP10D, and FADS1–3. Variants in 3 loci (ATP10D, FADS3, and SPTLC3) associate with MI in a series of three German MI studies. An additional 70 variants across 23 candidate genes involved in sphingolipid-metabolizing pathways also demonstrate association (p = 10−4 or less). Circulating concentrations of several key components in sphingolipid metabolism are thus under strong genetic control, and variants in these loci can be

  17. Introduction to Thematic Minireview Series: Novel Bioactive Sphingolipids.

    PubMed

    Merrill, Alfred H; Carman, George M

    2015-06-19

    Sphingosine was named by J. L. W. Thudichum for its enigmatic properties. This descriptor has applied to sphingolipids for over a century because new enigmas continue to surface. This JBC minireview series presents articles about three novel subspecies of sphingolipids, α-galactosylceramides, 4,5-dihydroceramides, and 1-deoxysphingolipids, that have important activities but, until recently, remained undetected (or at least understudied) in the shadow of very closely related compounds. They also serve as a reminder that important metabolites still lie "off the radar screen" in reports of global and comprehensive metabolomic profiling. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Everybody needs sphingolipids, right! Mining for new drug targets in protozoan sphingolipid biosynthesis.

    PubMed

    Mina, John G M; Denny, P W

    2017-06-22

    Sphingolipids (SLs) are an integral part of all eukaryotic cellular membranes. In addition, they have indispensable functions as signalling molecules controlling a myriad of cellular events. Disruption of either the de novo synthesis or the degradation pathways has been shown to have detrimental effects. The earlier identification of selective inhibitors of fungal SL biosynthesis promised potent broad-spectrum anti-fungal agents, which later encouraged testing some of those agents against protozoan parasites. In this review we focus on the key enzymes of the SL de novo biosynthetic pathway in protozoan parasites of the Apicomplexa and Kinetoplastidae, outlining the divergence and interconnection between host and pathogen metabolism. The druggability of the SL biosynthesis is considered, alongside recent technology advances that will enable the dissection and analyses of this pathway in the parasitic protozoa. The future impact of these advances for the development of new therapeutics for both globally threatening and neglected infectious diseases is potentially profound.

  19. Rice Stripe Virus Infection Alters mRNA Levels of Sphingolipid-Metabolizing Enzymes and Sphingolipids Content in Laodelphax striatellus

    PubMed Central

    Li, Fei-Qiang; Bai, Yue-Liang; Shi, Xiao-Xiao; Zhu, Mu-Fei; Zhang, Min-Jing; Mao, Cun-Gui; Zhu, Zeng-Rong

    2017-01-01

    Sphingolipids and their metabolites have been implicated in viral infection and replication in mammal cells but how their metabolizing enzymes in the host are regulated by viruses remains largely unknown. Here we report the identification of 12 sphingolipid genes and their regulation by Rice stripe virus in the small brown planthopper (Laodelphax striatellus Fallén), a serious pest of rice throughout eastern Asia. According to protein sequence similarity, we identified 12 sphingolipid enzyme genes in L. striatellus. By comparing their mRNA levels in viruliferous versus nonviruliferous L. striatellus at different life stages by qPCR, we found that RSV infection upregulated six genes (LsCGT1, LsNAGA1, LsSGPP, LsSMPD4, LsSMS, and LsSPT) in most stages of L. striatellus. Especially, four genes (LsCGT1, LsSMPD2, LsNAGA1, and LsSMS) and another three genes (LsNAGA1, LsSGPP, and LsSMS) were significantly upregulated in viruliferous third-instar and fourth-instar nymphs, respectively. HPLC-MS/MS results showed that RSV infection increased the levels of various ceramides, such as Cer18:0, Cer20:0, and Cer22:0 species, in third and fourth instar L. striatellus nymphs. Together, these results demonstrate that RSV infection alters the transcript levels of various sphingolipid enzymes and the contents of sphingolipids in L. striatellus, indicating that sphingolipids may be important for RSV infection or replication in L. striatellus. PMID:28130458

  20. Direct chemical evidence for sphingolipid domains in the plasma membranes of fibroblasts [High-Resolution Chemical Imaging of Sphingolipid Distribution in the Plasma Membrane

    SciTech Connect

    Frisz, Jessica F.; Lou, Kaiyan; Klitzing, Haley A.; Hanafin, William P.; Lizunov, Vladimir; Wilson, Robert L.; Carpenter, Kevin J.; Kim, Raehyun; Hutcheon, Ian D.; Zimmerberg, Joshua; Weber, Peter K.; Kraft, Mary L.

    2013-01-28

    Sphingolipids play important roles in plasma membrane structure and cell signaling. Yet, their lateral distribution in the plasma membrane is poorly understood. Here we quantitatively analyzed the sphingolipid organization on the entire dorsal surface of intact cells by mapping the distribution of 15N-enriched ions from metabolically labeled 15N-sphingolipids in the plasma membrane using high-resolution imaging mass spectrometry. Many types of control experiments (internal, positive, negative, and fixation temperature), along with parallel experiments involving the imaging of fluorescent sphingolipids$-$both in living cells and during fixation of living cells$-$exclude potential artifacts. Micrometer-scale sphingolipid patches consisting of numerous 15Nsphingolipid microdomains with mean diameters of ~200 nm are always present in the plasma membrane. Depletion of 30% of the cellular cholesterol did not eliminate the sphingolipid domains, but did reduce their abundance and long range organization in the plasma membrane. In contrast, disruption of the cytoskeleton eliminated the sphingolipid domains. These results indicate that these sphingolipid assemblages are not lipid rafts, and are instead a distinctly different type of sphingolipid-enriched plasma membrane domain that depends upon cortical actin.

  1. Direct chemical evidence for sphingolipid domains in the plasma membranes of fibroblasts [High-Resolution Chemical Imaging of Sphingolipid Distribution in the Plasma Membrane

    DOE PAGES

    Frisz, Jessica F.; Lou, Kaiyan; Klitzing, Haley A.; ...

    2013-01-28

    Sphingolipids play important roles in plasma membrane structure and cell signaling. Yet, their lateral distribution in the plasma membrane is poorly understood. Here we quantitatively analyzed the sphingolipid organization on the entire dorsal surface of intact cells by mapping the distribution of 15N-enriched ions from metabolically labeled 15N-sphingolipids in the plasma membrane using high-resolution imaging mass spectrometry. Many types of control experiments (internal, positive, negative, and fixation temperature), along with parallel experiments involving the imaging of fluorescent sphingolipids$-$both in living cells and during fixation of living cells$-$exclude potential artifacts. Micrometer-scale sphingolipid patches consisting of numerous 15Nsphingolipid microdomains with mean diametersmore » of ~200 nm are always present in the plasma membrane. Depletion of 30% of the cellular cholesterol did not eliminate the sphingolipid domains, but did reduce their abundance and long range organization in the plasma membrane. In contrast, disruption of the cytoskeleton eliminated the sphingolipid domains. These results indicate that these sphingolipid assemblages are not lipid rafts, and are instead a distinctly different type of sphingolipid-enriched plasma membrane domain that depends upon cortical actin.« less

  2. Two new sphingolipids from the leaves of Piper betle L.

    PubMed

    Chen, Duo-Zhi; Xiong, Hua-Bin; Tian, Kai; Guo, Jun-Ming; Huang, Xiang-Zhong; Jiang, Zhi-Yong

    2013-09-12

    Two new sphingolipids, pipercerebrosides A (1) and B (2), were isolated from the leaves of Piper betle L. Their structures, including absolute configurations, were determined by spectroscopic analysis and chemical degradation. These two compounds did not show significant cytotoxic activity against the cancer cell lines K562 and HL-60 in a MTT assay.

  3. Sphingolipid domains in the plasma membranes of fibroblasts are not enriched with cholesterol

    SciTech Connect

    Frisz, Jessica F.; Klitzing, Haley A.; Lou, Kaiyan; Hutcheon, Ian D.; Weber, Peter K.; Zimmerberg, Joshua; Kraft, Mary L.

    2013-04-22

    The plasma membranes of mammalian cells are widely expected to contain domains that are enriched with cholesterol and sphingolipids. In this work, we have used high-resolution secondary ion mass spectrometry to directly map the distributions of isotope-labeled cholesterol and sphingolipids in the plasma membranes of intact fibroblast cells. Although acute cholesterol depletion reduced sphingolipid domain abundance, cholesterol was evenly distributed throughout the plasma membrane and was not enriched within the sphingolipid domains. As a result, we rule out favorable cholesterol-sphingolipid interactions as dictating plasma membrane organization in fibroblast cells. Because the sphingolipid domains are disrupted by drugs that depolymerize the cells actin cytoskeleton, cholesterol must instead affect the sphingolipid organization via an indirect mechanism that involves the cytoskeleton.

  4. Metabolism, Physiological Role, and Clinical Implications of Sphingolipids in Gastrointestinal Tract

    PubMed Central

    Łukaszuk, Bartłomiej; Piotrowska, Dominika M.; Wiesiołek, Patrycja; Chabowska, Anna Małgorzata; Żendzian-Piotrowska, Małgorzata

    2013-01-01

    Sphingolipids in digestive system are responsible for numerous important physiological and pathological processes. In the membrane of gut epithelial cells, sphingolipids provide structural integrity, regulate absorption of some nutrients, and act as receptors for many microbial antigens and their toxins. Moreover, bioactive sphingolipids such as ceramide or sphingosine-1-phosphate regulate cellular growth, differentiation, and programmed cell death—apoptosis. Although it is well established that sphingolipids have clinical implications in gastrointestinal tumorigenesis or inflammation, further studies are needed to fully explore the role of sphingolipids in neoplastic and inflammatory diseases in gastrointestinal tract. Pharmacological agents which regulate metabolism of sphingolipids can be potentially used in the management of colorectal cancer or inflammatory bowel diseases. The aim of this work is to critically the review physiological and pathological roles of sphingolipids in the gastrointestinal tract. PMID:24083248

  5. Metabolism, physiological role, and clinical implications of sphingolipids in gastrointestinal tract.

    PubMed

    Kurek, Krzysztof; Łukaszuk, Bartłomiej; Piotrowska, Dominika M; Wiesiołek, Patrycja; Chabowska, Anna Małgorzata; Zendzian-Piotrowska, Małgorzata

    2013-01-01

    Sphingolipids in digestive system are responsible for numerous important physiological and pathological processes. In the membrane of gut epithelial cells, sphingolipids provide structural integrity, regulate absorption of some nutrients, and act as receptors for many microbial antigens and their toxins. Moreover, bioactive sphingolipids such as ceramide or sphingosine-1-phosphate regulate cellular growth, differentiation, and programmed cell death-apoptosis. Although it is well established that sphingolipids have clinical implications in gastrointestinal tumorigenesis or inflammation, further studies are needed to fully explore the role of sphingolipids in neoplastic and inflammatory diseases in gastrointestinal tract. Pharmacological agents which regulate metabolism of sphingolipids can be potentially used in the management of colorectal cancer or inflammatory bowel diseases. The aim of this work is to critically the review physiological and pathological roles of sphingolipids in the gastrointestinal tract.

  6. Sphingolipid domains in the plasma membranes of fibroblasts are not enriched with cholesterol

    DOE PAGES

    Frisz, Jessica F.; Klitzing, Haley A.; Lou, Kaiyan; ...

    2013-04-22

    The plasma membranes of mammalian cells are widely expected to contain domains that are enriched with cholesterol and sphingolipids. In this work, we have used high-resolution secondary ion mass spectrometry to directly map the distributions of isotope-labeled cholesterol and sphingolipids in the plasma membranes of intact fibroblast cells. Although acute cholesterol depletion reduced sphingolipid domain abundance, cholesterol was evenly distributed throughout the plasma membrane and was not enriched within the sphingolipid domains. As a result, we rule out favorable cholesterol-sphingolipid interactions as dictating plasma membrane organization in fibroblast cells. Because the sphingolipid domains are disrupted by drugs that depolymerize themore » cells actin cytoskeleton, cholesterol must instead affect the sphingolipid organization via an indirect mechanism that involves the cytoskeleton.« less

  7. Sphingolipid Domains in the Plasma Membranes of Fibroblasts Are Not Enriched with Cholesterol*

    PubMed Central

    Frisz, Jessica F.; Klitzing, Haley A.; Lou, Kaiyan; Hutcheon, Ian D.; Weber, Peter K.; Zimmerberg, Joshua; Kraft, Mary L.

    2013-01-01

    The plasma membranes of mammalian cells are widely expected to contain domains that are enriched with cholesterol and sphingolipids. In this work, we have used high-resolution secondary ion mass spectrometry to directly map the distributions of isotope-labeled cholesterol and sphingolipids in the plasma membranes of intact fibroblast cells. Although acute cholesterol depletion reduced sphingolipid domain abundance, cholesterol was evenly distributed throughout the plasma membrane and was not enriched within the sphingolipid domains. Thus, we rule out favorable cholesterol-sphingolipid interactions as dictating plasma membrane organization in fibroblast cells. Because the sphingolipid domains are disrupted by drugs that depolymerize the cells actin cytoskeleton, cholesterol must instead affect the sphingolipid organization via an indirect mechanism that involves the cytoskeleton. PMID:23609440

  8. Interictal, circulating sphingolipids in women with episodic migraine

    PubMed Central

    Mielke, Michelle M.; Dickens, Alex M.; Chatterjee, Subroto; Dash, Paul; Alexander, Guillermo; Vieira, Rebeca V.A.; Bandaru, Veera Venkata Ratnam; Dorskind, Joelle M.; Tietjen, Gretchen E.; Haughey, Norman H.

    2015-01-01

    Objective: To evaluate interictal, circulating sphingolipids in women migraineurs. Methods: In the fasting state, serum samples were obtained pain-free from 88 women with episodic migraine (EM; n = 52) and from controls (n = 36). Sphingolipids were detected and quantified by high-performance liquid chromatography coupled with tandem mass spectrometry using multiple reaction monitoring. Multivariate logistic regression was used to examine the association between serum sphingolipids and EM odds. A recursive partitioning decision tree based on the serum concentrations of 10 sphingolipids was used to determine the presence or absence of EM in a subset of participants. Results: Total ceramide (EM 6,502.9 ng/mL vs controls 10,518.5 ng/mL; p < 0.0001) and dihydroceramide (EM 39.3 ng/mL vs controls 63.1 ng/mL; p < 0.0001) levels were decreased in those with EM as compared with controls. Using multivariate logistic regression, each SD increase in total ceramide (odds ratio [OR] 0.07; 95% confidence interval [CI]: 0.02, 0.22; p < 0.001) and total dihydroceramide (OR 0.05; 95% CI: 0.01, 0.21; p < 0.001) levels was associated with more than 92% reduced odds of migraine. Although crude sphingomyelin levels were not different in EM compared with controls, after adjustments, every SD increase in the sphingomyelin species C18:0 (OR 4.28; 95% CI: 1.87, 9.81; p = 0.001) and C18:1 (OR 2.93; 95% CI: 1.55, 5.54; p = 0.001) was associated with an increased odds of migraine. Recursive portioning models correctly classified 14 of 14 randomly selected participants as EM or control. Conclusion: These results suggest that sphingolipid metabolism is altered in women with EM and that serum sphingolipid panels may have potential to differentiate EM presence or absence. Classification of evidence: This study provides Class III evidence that serum sphingolipid panels accurately distinguish women with migraine from women without migraine. PMID:26354990

  9. An overview of sphingolipid metabolism: from synthesis to breakdown

    PubMed Central

    Gault, CR; Obeid, LM; Hannun, YA

    2011-01-01

    Sphingolipids constitute a class of lipids defined by their eighteen carbon amino-alcohol backbones which are synthesized in the ER from nonsphingolipid precursors. Modification of this basic structure is what gives rise to the vast family of sphingolipids that play significant roles in membrane biology and provide many bioactive metabolites that regulate cell function. Despite the diversity of structure and function of sphingolipids, their creation and destruction are governed by common synthetic and catabolic pathways. In this regard, sphingolipid metabolism can be imagined as an array of interconnected networks that diverge from a single common entry point and converge into a single common breakdown pathway. In their simplest forms, sphingosine, phytosphingosine and dihydrosphingosine serve as the backbones upon which further complexity is achieved. For example, phosphorylation of the C1 hydroxyl group yields the final breakdown products and/or the important signaling molecules sphingosine-1-phosphate, phytosphingosine-1-phosphate and dihydrosphingosine-1-phosphate, respectively. On the other hand, acylation of sphingosine, phytosphingosine, or dihydrosphingosine with one of several possible acyl CoA molecules through the action of distinct ceramide synthases produces the molecules defined as ceramide, phytoceramide, or dihydroceramide. Ceramide, due to the differing acyl CoAs that can be used to produce it, is technically a class of molecules rather than a single molecule and therefore may have different biological functions depending on the acyl chain it is composed of. At the apex of complexity is the group of lipids known as glycosphingolipids (GSL) which contain dozens of different sphingolipid species differing by both the order and type of sugar residues attached to their headgroups. Since these molecules are produced from ceramide precursors, they too may have differences in their acyl chain composition, revealing an additional layer of variation. The

  10. Sphingolipid modulation of angiogenic factor expression in neuroblastoma

    PubMed Central

    Li, Mei-Hong; Hla, Timothy; Ferrer, Fernando

    2011-01-01

    Metabolism of sphingolipids into downstream lipid mediators followed by signaling modulates tumor microenvironment and the cancer cells to influence tumor progression. As such, sphingolipid signaling represents a novel way to modulate tumor biology. Neuroblastoma (NB), the most common extracranial solid tumor of childhood, is highly angiogenic and often displays poor prognosis. However, the role of sphingolipid mediators is not known in NB. We found that NB expresses high levels of sphingosine kinase-2 (SphK2), which is essential for the formation of sphingosine-1-phosphate (S1P). S1P induced vascular endothelial growth factor (VEGF) expression in SK-N-AS NB cells. The effect occurred at the transcriptional level. Hypoxia in combination with S1P had a synergistic effect on VEGF expression. Strong correlation was detected between S1P receptor-2 (S1P2) and VEGF mRNAs in 11 different cell lines and 17 NB tissues. Blockade of S1P2 with the selective antagonist JTE-013 significantly inhibited S1P-induced VEGF expression. Overexpression and knockdown of S1P2 in SK-N-AS cells increased or inhibited S1P-induced VEGF secretion, respectively. Interestingly, JTE-013 significantly inhibited tumor growth, VEGF mRNA expression and induced apoptosis in the NB tumor xenografts. Taken together, our data suggest that enhanced formation of sphingolipid mediator S1P in NB profoundly influences tumor microenvironment by inducing VEGF expression via S1P2. Modulation of sphingolipid signaling by inhibiting S1P2 may constitute a novel strategy to control NB. PMID:21576349

  11. Runx1 Orchestrates Sphingolipid Metabolism and Glucocorticoid Resistance in Lymphomagenesis

    PubMed Central

    Terry, A.; Wotton, S.; Borland, G.; Zhang, Q.; Mackay, N.; McDonald, A.; Bell, M.; Wakelam, M.J.O.; Cameron, E.R.

    2017-01-01

    ABSTRACT The three‐membered RUNX gene family includes RUNX1, a major mutational target in human leukemias, and displays hallmarks of both tumor suppressors and oncogenes. In mouse models, the Runx genes appear to act as conditional oncogenes, as ectopic expression is growth suppressive in normal cells but drives lymphoma development potently when combined with over‐expressed Myc or loss of p53. Clues to underlying mechanisms emerged previously from murine fibroblasts where ectopic expression of any of the Runx genes promotes survival through direct and indirect regulation of key enzymes in sphingolipid metabolism associated with a shift in the “sphingolipid rheostat” from ceramide to sphingosine‐1‐phosphate (S1P). Testing of this relationship in lymphoma cells was therefore a high priority. We find that ectopic expression of Runx1 in lymphoma cells consistently perturbs the sphingolipid rheostat, whereas an essential physiological role for Runx1 is revealed by reduced S1P levels in normal spleen after partial Cre‐mediated excision. Furthermore, we show that ectopic Runx1 expression confers increased resistance of lymphoma cells to glucocorticoid‐mediated apoptosis, and elucidate the mechanism of cross‐talk between glucocorticoid and sphingolipid metabolism through Sgpp1. Dexamethasone potently induces expression of Sgpp1 in T‐lymphoma cells and drives cell death which is reduced by partial knockdown of Sgpp1 with shRNA or direct transcriptional repression of Sgpp1 by ectopic Runx1. Together these data show that Runx1 plays a role in regulating the sphingolipid rheostat in normal development and that perturbation of this cell fate regulator contributes to Runx‐driven lymphomagenesis. J. Cell. Biochem. 118: 1432–1441, 2017. © 2016 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc. PMID:27869314

  12. Runx1 Orchestrates Sphingolipid Metabolism and Glucocorticoid Resistance in Lymphomagenesis.

    PubMed

    Kilbey, A; Terry, A; Wotton, S; Borland, G; Zhang, Q; Mackay, N; McDonald, A; Bell, M; Wakelam, M J O; Cameron, E R; Neil, J C

    2017-06-01

    The three-membered RUNX gene family includes RUNX1, a major mutational target in human leukemias, and displays hallmarks of both tumor suppressors and oncogenes. In mouse models, the Runx genes appear to act as conditional oncogenes, as ectopic expression is growth suppressive in normal cells but drives lymphoma development potently when combined with over-expressed Myc or loss of p53. Clues to underlying mechanisms emerged previously from murine fibroblasts where ectopic expression of any of the Runx genes promotes survival through direct and indirect regulation of key enzymes in sphingolipid metabolism associated with a shift in the "sphingolipid rheostat" from ceramide to sphingosine-1-phosphate (S1P). Testing of this relationship in lymphoma cells was therefore a high priority. We find that ectopic expression of Runx1 in lymphoma cells consistently perturbs the sphingolipid rheostat, whereas an essential physiological role for Runx1 is revealed by reduced S1P levels in normal spleen after partial Cre-mediated excision. Furthermore, we show that ectopic Runx1 expression confers increased resistance of lymphoma cells to glucocorticoid-mediated apoptosis, and elucidate the mechanism of cross-talk between glucocorticoid and sphingolipid metabolism through Sgpp1. Dexamethasone potently induces expression of Sgpp1 in T-lymphoma cells and drives cell death which is reduced by partial knockdown of Sgpp1 with shRNA or direct transcriptional repression of Sgpp1 by ectopic Runx1. Together these data show that Runx1 plays a role in regulating the sphingolipid rheostat in normal development and that perturbation of this cell fate regulator contributes to Runx-driven lymphomagenesis. J. Cell. Biochem. 118: 1432-1441, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  13. Regulation of PP2A by Sphingolipid Metabolism and Signaling

    PubMed Central

    Oaks, Joshua; Ogretmen, Besim

    2014-01-01

    Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase that is a primary regulator of cellular proliferation through targeting of proliferative kinases, cell cycle regulators, and apoptosis inhibitors. It is through the regulation of these regulatory elements that gives PP2A tumor suppressor functions. In addition to mutations on the regulatory subunits, the phosphatase/tumor suppressing activity of PP2A is also inhibited in several cancer types due to overexpression or modification of the endogenous PP2A inhibitors such as SET/I2PP2A. This review focuses on the current literature regarding the interactions between the lipid signaling molecules, selectively sphingolipids, and the PP2A inhibitor SET for the regulation of PP2A, and the therapeutic potential of sphingolipids as PP2A activators for tumor suppression via targeting SET oncoprotein. PMID:25642418

  14. Regulation of PP2A by Sphingolipid Metabolism and Signaling.

    PubMed

    Oaks, Joshua; Ogretmen, Besim

    2014-01-01

    Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase that is a primary regulator of cellular proliferation through targeting of proliferative kinases, cell cycle regulators, and apoptosis inhibitors. It is through the regulation of these regulatory elements that gives PP2A tumor suppressor functions. In addition to mutations on the regulatory subunits, the phosphatase/tumor suppressing activity of PP2A is also inhibited in several cancer types due to overexpression or modification of the endogenous PP2A inhibitors such as SET/I2PP2A. This review focuses on the current literature regarding the interactions between the lipid signaling molecules, selectively sphingolipids, and the PP2A inhibitor SET for the regulation of PP2A, and the therapeutic potential of sphingolipids as PP2A activators for tumor suppression via targeting SET oncoprotein.

  15. Sphingolipids contribute to acetic acid resistance in Zygosaccharomyces bailii.

    PubMed

    Lindahl, Lina; Genheden, Samuel; Eriksson, Leif A; Olsson, Lisbeth; Bettiga, Maurizio

    2016-04-01

    Lignocellulosic raw material plays a crucial role in the development of sustainable processes for the production of fuels and chemicals. Weak acids such as acetic acid and formic acid are troublesome inhibitors restricting efficient microbial conversion of the biomass to desired products. To improve our understanding of weak acid inhibition and to identify engineering strategies to reduce acetic acid toxicity, the highly acetic-acid-tolerant yeast Zygosaccharomyces bailii was studied. The impact of acetic acid membrane permeability on acetic acid tolerance in Z. bailii was investigated with particular focus on how the previously demonstrated high sphingolipid content in the plasma membrane influences acetic acid tolerance and membrane permeability. Through molecular dynamics simulations, we concluded that membranes with a high content of sphingolipids are thicker and more dense, increasing the free energy barrier for the permeation of acetic acid through the membrane. Z. bailii cultured with the drug myriocin, known to decrease cellular sphingo-lipid levels, exhibited significant growth inhibition in the presence of acetic acid, while growth in medium without acetic acid was unaffected by the myriocin addition. Furthermore, following an acetic acid pulse, the intracellular pH decreased more in myriocin-treated cells than in control cells. This indicates a higher inflow rate of acetic acid and confirms that the reduction in growth of cells cultured with myriocin in the medium with acetic acid was due to an increase in membrane permeability, thereby demonstrating the importance of a high fraction of sphingolipids in the membrane of Z. bailii to facilitate acetic acid resistance; a property potentially transferable to desired production organisms suffering from weak acid stress. © 2015 Wiley Periodicals, Inc.

  16. Beyond the Cherry-Red Spot: Ocular Manifestations of Sphingolipid-mediated Neurodegenerative and Inflammatory Disorders

    PubMed Central

    Stone, Donald U.; Mandal, Nawajes A.

    2013-01-01

    Sphingolipids are a ubiquitous membrane lipid present in every cell and found most abundantly in neural tissues. Disorders such as Tay Sachs or Niemann Pick disease are the most familiar examples of dysfunction in sphingolipid metabolism and are typically associated with neurodegeneration and ocular findings such as blindness. More recently, the role of bioactive sphingolipids has been established in a multitude of cellular events, including cell survival, growth, senescence and apoptosis, inflammation, and neovascularization. We discuss our current knowledge and understanding of sphingolipid metabolism and signaling in the pathogenesis of ocular diseases. PMID:24011710

  17. Genes encoding Δ(8)-sphingolipid desaturase from various plants: identification, biochemical functions, and evolution.

    PubMed

    Li, Shu-Fen; Zhang, Guo-Jun; Zhang, Xue-Jin; Yuan, Jin-Hong; Deng, Chuan-Liang; Hu, Zan-Min; Gao, Wu-Jun

    2016-09-01

    ∆(8)-sphingolipid desaturase catalyzes the C8 desaturation of a long chain base, which is the characteristic structure of various complex sphingolipids. The genes of 20 ∆(8)-sphingolipid desaturases from 12 plants were identified and functionally detected by using Saccharomyces cerevisiae system to elucidate the relationship between the biochemical function and evolution of this enzyme. Results showed that the 20 genes all can encode a functional ∆(8)-sphingolipid desaturase, which catalyzes different ratios of two products, namely, 8(Z) and 8(E)-C18-phytosphingenine. The coded enzymes could be divided into two groups on the basis of biochemical functions: ∆(8)-sphingolipid desaturase with a preference for an E-isomer product and ∆(8)-sphingolipid desaturase with a preference for a Z-isomer product. The conversion rate of the latter was generally lower than that of the former. Phylogenetic analysis revealed that the 20 desaturases could also be clustered into two groups, and this grouping is consistent with that of the biochemical functions. Thus, the biochemical function of ∆(8)-sphingolipid desaturase is correlated with its evolution. The two groups of ∆(8)-sphingolipid desaturases could arise from distinct ancestors in higher plants. However, they might have initially evolved from ∆(8)-sphingolipid desaturases in lower organisms, such as yeasts, which can produce E-isomer products only. Furthermore, almost all of the transgenic yeasts harboring ∆(8)-sphingolipid desaturase genes exhibit an improvement in aluminum tolerance. Our study provided new insights into the biochemical function and evolution of ∆(8)-sphingolipid desaturases in plants.

  18. Harnessing the power of yeast to elucidate the role of sphingolipids in metabolic and signaling processes pertinent to psychiatric disorders

    PubMed Central

    Jadhav, Shyamalagauri; Greenberg, Miriam L

    2015-01-01

    The development of therapies for neuropsychiatric disorders is hampered by the lack of understanding of the mechanisms underlying their pathologies. While aberrant sphingolipid metabolism is associated with psychiatric illness, the role of sphingolipids in these disorders is not understood. The genetically tractable yeast model can be exploited in order to elucidate the cellular consequences of sphingolipid perturbation. Hypotheses generated from studies in yeast and tested in mammalian cells may contribute to our understanding of the role of sphingolipids in psychiatric disorders and to the development of new treatments. Here, we compare sphingolipid metabolism in yeast and mammalian cells, discuss studies implicating sphingolipids in psychiatric disorders and propose approaches that utilize yeast in order to elucidate sphingolipid function and identify drugs that target sphingolipid synthesis. PMID:25750665

  19. Sphingolipids contribute to acetic acid resistance in Zygosaccharomyces bailii

    PubMed Central

    Lindahl, Lina; Genheden, Samuel; Eriksson, Leif A.; Olsson, Lisbeth

    2015-01-01

    ABSTRACT Lignocellulosic raw material plays a crucial role in the development of sustainable processes for the production of fuels and chemicals. Weak acids such as acetic acid and formic acid are troublesome inhibitors restricting efficient microbial conversion of the biomass to desired products. To improve our understanding of weak acid inhibition and to identify engineering strategies to reduce acetic acid toxicity, the highly acetic‐acid‐tolerant yeast Zygosaccharomyces bailii was studied. The impact of acetic acid membrane permeability on acetic acid tolerance in Z. bailii was investigated with particular focus on how the previously demonstrated high sphingolipid content in the plasma membrane influences acetic acid tolerance and membrane permeability. Through molecular dynamics simulations, we concluded that membranes with a high content of sphingolipids are thicker and more dense, increasing the free energy barrier for the permeation of acetic acid through the membrane. Z. bailii cultured with the drug myriocin, known to decrease cellular sphingo­lipid levels, exhibited significant growth inhibition in the presence of acetic acid, while growth in medium without acetic acid was unaffected by the myriocin addition. Furthermore, following an acetic acid pulse, the intracellular pH decreased more in myriocin‐treated cells than in control cells. This indicates a higher inflow rate of acetic acid and confirms that the reduction in growth of cells cultured with myriocin in the medium with acetic acid was due to an increase in membrane permeability, thereby demonstrating the importance of a high fraction of sphingolipids in the membrane of Z. bailii to facilitate acetic acid resistance; a property potentially transferable to desired production organisms suffering from weak acid stress. Biotechnol. Bioeng. 2016;113: 744–753. © 2015 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. PMID:26416641

  20. Characteristics of the Rat Cardiac Sphingolipid Pool in Two Mitochondrial Subpopulations

    PubMed Central

    Monette, Jeffrey S.; Gómez, Luis A.; Moreau, Régis F.; Bemer, Brett A.; Taylor, Alan W.; Hagen, Tory M.

    2010-01-01

    Mitochondrial sphingolipids play a diverse role in normal cardiac function and diseases, yet a precise quantification of cardiac mitochondrial sphingolipids has never been performed. Therefore, rat heart interfibrillary (IFM) and subsarcolemmal (SSM) mitochondria were isolated, lipids extracted, and sphingolipids quantified by LC-tandem mass spectrometry. Results showed that sphingomyelin (~10,000 pmols/mg protein) was the predominant sphingolipid regardless of mitochondrial subpopulation, and measurable amounts of ceramide (~70 pmols/mg protein) sphingosine, and sphinganine were also found in IFM and SSM. Both mitochondrial populations contained similar quantities of sphingolipids except for ceramide which was much higher in SSM. Analysis of sphingolipid isoforms revealed ten different sphingomyelins and six ceramides that differed from 16 to 24 carbon units in their acyl side-chains. Sub-fractionation experiments further showed that sphingolipids are a constituent part of the inner mitochondrial membrane. Furthermore, inner membrane ceramide levels were 32% lower versus whole mitochondria (45 pmols/mg protein). Three ceramide isotypes (C20-, C22-, and C24-ceramide) accounted for the lower amounts. The concentrations of the ceramides present in the inner membranes of SSM and IFM differed greatly. Overall, mitochondrial sphingolipid content reflected levels seen in cardiac tissue, but the specific ceramide distribution distinguished IFM and SSM from each other. PMID:20599536

  1. Characteristics of the rat cardiac sphingolipid pool in two mitochondrial subpopulations.

    PubMed

    Monette, Jeffrey S; Gómez, Luis A; Moreau, Régis F; Bemer, Brett A; Taylor, Alan W; Hagen, Tory M

    2010-07-23

    Mitochondrial sphingolipids play a diverse role in normal cardiac function and diseases, yet a precise quantification of cardiac mitochondrial sphingolipids has never been performed. Therefore, rat heart interfibrillary mitochondria (IFM) and subsarcolemmal mitochondria (SSM) were isolated, lipids extracted, and sphingolipids quantified by LC-tandem mass spectrometry. Results showed that sphingomyelin (approximately 10,000 pmol/mg protein) was the predominant sphingolipid regardless of mitochondrial subpopulation, and measurable amounts of ceramide (approximately 70 pmol/mg protein) sphingosine, and sphinganine were also found in IFM and SSM. Both mitochondrial populations contained similar quantities of sphingolipids except for ceramide which was much higher in SSM. Analysis of sphingolipid isoforms revealed ten different sphingomyelins and six ceramides that differed from 16- to 24-carbon units in their acyl side chains. Sub-fractionation experiments further showed that sphingolipids are a constituent part of the inner mitochondrial membrane. Furthermore, inner membrane ceramide levels were 32% lower versus whole mitochondria (45 pmol/mg protein). Three ceramide isotypes (C20-, C22-, and C24-ceramide) accounted for the lower amounts. The concentrations of the ceramides present in the inner membranes of SSM and IFM differed greatly. Overall, mitochondrial sphingolipid content reflected levels seen in cardiac tissue, but the specific ceramide distribution distinguished IFM and SSM from each other.

  2. A systematic simulation of the effect of salicylic acid on sphingolipid metabolism

    PubMed Central

    Shi, Chao; Yin, Jian; Liu, Zhe; Wu, Jian-Xin; Zhao, Qi; Ren, Jian; Yao, Nan

    2015-01-01

    The phytohormone salicylic acid (SA) affects plant development and defense responses. Recent studies revealed that SA also participates in the regulation of sphingolipid metabolism, but the details of this regulation remain to beexplored. Here, we use in silico Flux Balance Analysis (FBA) with published microarray data to construct a whole-cell simulation model, including 23 pathways, 259 reactions, and 172 metabolites, to predict the alterations in flux of major sphingolipid species after treatment with exogenous SA. This model predicts significant changes in fluxes of certain sphingolipid species after SA treatment, changes that likely trigger downstream physiological and phenotypic effects. To validate the simulation, we used 15N-labeled metabolic turnover analysis to measure sphingolipid contents and turnover rate in Arabidopsis thaliana seedlings treated with SA or the SA analog benzothiadiazole (BTH). The results show that both SA and BTH affect sphingolipid metabolism, altering the concentrations of certain species and also changing the optimal flux distribution and turnover rate of sphingolipids. Our strategy allows us to estimate sphingolipid fluxes on a short time scale and gives us a systemic view of the effect of SA on sphingolipid homeostasis. PMID:25859253

  3. Sphingolipid regulation of ezrin, radixin, and moesin proteins family: implications for cell dynamics.

    PubMed

    Adada, Mohamad; Canals, Daniel; Hannun, Yusuf A; Obeid, Lina M

    2014-05-01

    A key but poorly studied domain of sphingolipid functions encompasses endocytosis, exocytosis, cellular trafficking, and cell movement. Recently, the ezrin, radixin and moesin (ERM) family of proteins emerged as novel potent targets regulated by sphingolipids. ERMs are structural proteins linking the actin cytoskeleton to the plasma membrane, also forming a scaffold for signaling pathways that are used for cell proliferation, migration and invasion, and cell division. Opposing functions of the bioactive sphingolipid ceramide and sphingosine-1-phosphate (S1P), contribute to ERM regulation. S1P robustly activates whereas ceramide potently deactivates ERM via phosphorylation/dephosphorylation, respectively. This recent dimension of cytoskeletal regulation by sphingolipids opens up new avenues to target cell dynamics, and provides further understanding of some of the unexplained biological effects mediated by sphingolipids. In addition, these studies are providing novel inroads into defining basic mechanisms of regulation and action of bioactive sphingolipids. This review describes the current understanding of sphingolipid regulation of the cytoskeleton, it also describes the biologies in which ERM proteins have been involved, and finally how these two large fields have started to converge. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.

  4. GIPC: Glycosyl Inositol Phospho Ceramides, the major sphingolipids on earth.

    PubMed

    Gronnier, Julien; Germain, Véronique; Gouguet, Paul; Cacas, Jean-Luc; Mongrand, Sébastien

    2016-01-01

    What are the most abundant sphingolipids on earth? The answer is Glycosyl Inositol Phosphoryl Ceramides (GIPCs) present in fungi and the green lineage. In this review, we discuss the putative role of plant GIPCs in the lipid bilayer asymmetry, in the lateral organization of membrane rafts and in the very long chain fatty acid inter-leaflet coupling of lipids in the plant plasma membrane (PM). A special focus on the structural similarities -and putative functions- of GIPCs is discussed by comparison with animal gangliosides, structural homologs of plant GIPCs.

  5. GIPC: Glycosyl Inositol Phospho Ceramides, the major sphingolipids on earth

    PubMed Central

    Gronnier, Julien; Germain, Véronique; Gouguet, Paul; Cacas, Jean-Luc; Mongrand, Sébastien

    2016-01-01

    ABSTRACT What are the most abundant sphingolipids on earth? The answer is Glycosyl Inositol Phosphoryl Ceramides (GIPCs) present in fungi and the green lineage. In this review, we discuss the putative role of plant GIPCs in the lipid bilayer asymmetry, in the lateral organization of membrane rafts and in the very long chain fatty acid inter-leaflet coupling of lipids in the plant plasma membrane (PM). A special focus on the structural similarities -and putative functions- of GIPCs is discussed by comparison with animal gangliosides, structural homologs of plant GIPCs. PMID:27074617

  6. Hemagglutinin Clusters in the Plasma Membrane Are Not Enriched with Cholesterol and Sphingolipids

    DOE PAGES

    Wilson, Robert L.; Frisz, Jessica F.; Klitzing, Haley A.; ...

    2015-04-07

    The clusters of the influenza envelope protein, hemagglutinin, within the plasma membrane are hypothesized to be enriched with cholesterol and sphingolipids. Here in this paper, we directly tested this hypothesis by using high-resolution secondary ion mass spectrometry to image the distributions of antibody-labeled hemagglutinin and isotope-labeled cholesterol and sphingolipids in the plasma membranes of fibroblast cells that stably express hemagglutinin. We found that the hemagglutinin clusters were neither enriched with cholesterol nor colocalized with sphingolipid domains. Thus, hemagglutinin clustering and localization in the plasma membrane is not controlled by cohesive interactions between hemagglutinin and liquid-ordered domains enriched with cholesterol andmore » sphingolipids, or from specific binding interactions between hemagglutinin, cholesterol, and/or the majority of sphingolipid species in the plasma membrane.« less

  7. Sphingolipids regulate telomere clustering by affecting the transcription of genes involved in telomere homeostasis.

    PubMed

    Ikeda, Atsuko; Muneoka, Tetsuya; Murakami, Suguru; Hirota, Ayaka; Yabuki, Yukari; Karashima, Takefumi; Nakazono, Kota; Tsuruno, Masahiro; Pichler, Harald; Shirahige, Katsuhiko; Kodama, Yukiko; Shimamoto, Toshi; Mizuta, Keiko; Funato, Kouichi

    2015-07-15

    In eukaryotic organisms, including mammals, nematodes and yeasts, the ends of chromosomes, telomeres are clustered at the nuclear periphery. Telomere clustering is assumed to be functionally important because proper organization of chromosomes is necessary for proper genome function and stability. However, the mechanisms and physiological roles of telomere clustering remain poorly understood. In this study, we demonstrate a role for sphingolipids in telomere clustering in the budding yeast Saccharomyces cerevisiae. Because abnormal sphingolipid metabolism causes downregulation of expression levels of genes involved in telomere organization, sphingolipids appear to control telomere clustering at the transcriptional level. In addition, the data presented here provide evidence that telomere clustering is required to protect chromosome ends from DNA-damage checkpoint signaling. As sphingolipids are found in all eukaryotes, we speculate that sphingolipid-based regulation of telomere clustering and the protective role of telomere clusters in maintaining genome stability might be conserved in eukaryotes.

  8. Hemagglutinin Clusters in the Plasma Membrane Are Not Enriched with Cholesterol and Sphingolipids

    PubMed Central

    Wilson, Robert L.; Frisz, Jessica F.; Klitzing, Haley A.; Zimmerberg, Joshua; Weber, Peter K.; Kraft, Mary L.

    2015-01-01

    The clusters of the influenza envelope protein, hemagglutinin, within the plasma membrane are hypothesized to be enriched with cholesterol and sphingolipids. Here, we directly tested this hypothesis by using high-resolution secondary ion mass spectrometry to image the distributions of antibody-labeled hemagglutinin and isotope-labeled cholesterol and sphingolipids in the plasma membranes of fibroblast cells that stably express hemagglutinin. We found that the hemagglutinin clusters were neither enriched with cholesterol nor colocalized with sphingolipid domains. Thus, hemagglutinin clustering and localization in the plasma membrane is not controlled by cohesive interactions between hemagglutinin and liquid-ordered domains enriched with cholesterol and sphingolipids, or from specific binding interactions between hemagglutinin, cholesterol, and/or the majority of sphingolipid species in the plasma membrane. PMID:25863057

  9. Sphingolipid Organization in the Plasma Membrane and the Mechanisms That Influence It.

    PubMed

    Kraft, Mary L

    2016-01-01

    Sphingolipids are structural components in the plasma membranes of eukaryotic cells. Their metabolism produces bioactive signaling molecules that modulate fundamental cellular processes. The segregation of sphingolipids into distinct membrane domains is likely essential for cellular function. This review presents the early studies of sphingolipid distribution in the plasma membranes of mammalian cells that shaped the most popular current model of plasma membrane organization. The results of traditional imaging studies of sphingolipid distribution in stimulated and resting cells are described. These data are compared with recent results obtained with advanced imaging techniques, including super-resolution fluorescence detection and high-resolution secondary ion mass spectrometry (SIMS). Emphasis is placed on the new insight into the sphingolipid organization within the plasma membrane that has resulted from the direct imaging of stable isotope-labeled lipids in actual cell membranes with high-resolution SIMS. Super-resolution fluorescence techniques have recently revealed the biophysical behaviors of sphingolipids and the unhindered diffusion of cholesterol analogs in the membranes of living cells are ultimately in contrast to the prevailing hypothetical model of plasma membrane organization. High-resolution SIMS studies also conflicted with the prevailing hypothesis, showing sphingolipids are concentrated in micrometer-scale membrane domains, but cholesterol is evenly distributed within the plasma membrane. Reductions in cellular cholesterol decreased the number of sphingolipid domains in the plasma membrane, whereas disruption of the cytoskeleton eliminated them. In addition, hemagglutinin, a transmembrane protein that is thought to be a putative raft marker, did not cluster within sphingolipid-enriched regions in the plasma membrane. Thus, sphingolipid distribution in the plasma membrane is dependent on the cytoskeleton, but not on favorable interactions with

  10. Sphingolipid Organization in the Plasma Membrane and the Mechanisms That Influence It

    PubMed Central

    Kraft, Mary L.

    2017-01-01

    Sphingolipids are structural components in the plasma membranes of eukaryotic cells. Their metabolism produces bioactive signaling molecules that modulate fundamental cellular processes. The segregation of sphingolipids into distinct membrane domains is likely essential for cellular function. This review presents the early studies of sphingolipid distribution in the plasma membranes of mammalian cells that shaped the most popular current model of plasma membrane organization. The results of traditional imaging studies of sphingolipid distribution in stimulated and resting cells are described. These data are compared with recent results obtained with advanced imaging techniques, including super-resolution fluorescence detection and high-resolution secondary ion mass spectrometry (SIMS). Emphasis is placed on the new insight into the sphingolipid organization within the plasma membrane that has resulted from the direct imaging of stable isotope-labeled lipids in actual cell membranes with high-resolution SIMS. Super-resolution fluorescence techniques have recently revealed the biophysical behaviors of sphingolipids and the unhindered diffusion of cholesterol analogs in the membranes of living cells are ultimately in contrast to the prevailing hypothetical model of plasma membrane organization. High-resolution SIMS studies also conflicted with the prevailing hypothesis, showing sphingolipids are concentrated in micrometer-scale membrane domains, but cholesterol is evenly distributed within the plasma membrane. Reductions in cellular cholesterol decreased the number of sphingolipid domains in the plasma membrane, whereas disruption of the cytoskeleton eliminated them. In addition, hemagglutinin, a transmembrane protein that is thought to be a putative raft marker, did not cluster within sphingolipid-enriched regions in the plasma membrane. Thus, sphingolipid distribution in the plasma membrane is dependent on the cytoskeleton, but not on favorable interactions with

  11. Sphingolipids and mitochondrial function, lessons learned from yeast

    PubMed Central

    Spincemaille, Pieter; Cammue, Bruno P.; Thevissen, Karin

    2014-01-01

    Mitochondrial dysfunction is a hallmark of several neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease, but also of cancer, diabetes and rare diseases such as Wilson’s disease (WD) and Niemann Pick type C1 (NPC). Mitochondrial dysfunction underlying human pathologies has often been associated with an aberrant cellular sphingolipid metabolism. Sphingolipids (SLs) are important membrane constituents that also act as signaling molecules. The yeast Saccharomyces cerevisiae has been pivotal in unraveling mammalian SL metabolism, mainly due to the high degree of conservation of SL metabolic pathways. In this review we will first provide a brief overview of the major differences in SL metabolism between yeast and mammalian cells and the use of SL biosynthetic inhibitors to elucidate the contribution of specific parts of the SL metabolic pathway in response to for instance stress. Next, we will discuss recent findings in yeast SL research concerning a crucial signaling role for SLs in orchestrating mitochondrial function, and translate these findings to relevant disease settings such as WD and NPC. In summary, recent research shows that S. cerevisiae is an invaluable model to investigate SLs as signaling molecules in modulating mitochondrial function, but can also be used as a tool to further enhance our current knowledge on SLs and mitochondria in mammalian cells. PMID:28357246

  12. Sphingolipids: A Potential Molecular Approach to Treat Allergic Inflammation

    PubMed Central

    Sun, Wai Y.; Bonder, Claudine S.

    2012-01-01

    Allergic inflammation is an immune response to foreign antigens, which begins within minutes of exposure to the allergen followed by a late phase leading to chronic inflammation. Prolonged allergic inflammation manifests in diseases such as urticaria and rhino-conjunctivitis, as well as chronic asthma and life-threatening anaphylaxis. The prevalence of allergic diseases is profound with 25% of the worldwide population affected and a rising trend across all ages, gender, and racial groups. The identification and avoidance of allergens can manage this disease, but this is not always possible with triggers being common foods, prevalent air-borne particles and only extremely low levels of allergen exposure required for sensitization. Patients who are sensitive to multiple allergens require prophylactic and symptomatic treatments. Current treatments are often suboptimal and associated with adverse effects, such as the interruption of cognition, sleep cycles, and endocrine homeostasis, all of which affect quality of life and are a financial burden to society. Clearly, a better therapeutic approach for allergic diseases is required. Herein, we review the current knowledge of allergic inflammation and discuss the role of sphingolipids as potential targets to regulate inflammatory development in vivo and in humans. We also discuss the benefits and risks of using sphingolipid inhibitors. PMID:23316248

  13. Quantitative profiling of sphingolipids in wild Cordyceps and its mycelia by using UHPLC-MS.

    PubMed

    Mi, Jia-Ning; Wang, Jing-Rong; Jiang, Zhi-Hong

    2016-02-12

    In the present study, 101 sphingolipids in wild Cordyceps and its five mycelia were quantitatively profiled by using a fully validated UHPLC-MS method. The results revealed that a general rank order for the abundance of different classes of sphingolipids in wild Cordyceps and its mycelia is sphingoid bases/ceramides > phosphosphingolipids > glycosphingolipids. However, remarkable sphingolipid differences between wild Cordyceps and its mycelia were observed. One is that sphingoid base is the dominant sphingolipid in wild Cordyceps, whereas ceramide is the major sphingolipid in mycelia. Another difference is that the abundance of sphingomyelins in wild Cordyceps is almost 10-folds higher than those in most mycelia. The third one is that mycelia contain more inositol phosphorylceramides and glycosphingolipids than wild Cordyceps. Multivariate analysis was further employed to visualize the difference among wild Cordyceps and different mycelia, leading to the identification of respective sphingolipids as potential chemical markers for the differentiation of wild Cordyceps and its related mycelia. This study represents the first report on the quantitative profiling of sphingolipids in wild Cordyceps and its related mycelia, which provided comprehensive chemical evidence for the quality control and rational utilization of wild Cordyceps and its mycelia.

  14. Quantitative profiling of sphingolipids in wild Cordyceps and its mycelia by using UHPLC-MS

    PubMed Central

    Mi, Jia-Ning; Wang, Jing-Rong; Jiang, Zhi-Hong

    2016-01-01

    In the present study, 101 sphingolipids in wild Cordyceps and its five mycelia were quantitatively profiled by using a fully validated UHPLC-MS method. The results revealed that a general rank order for the abundance of different classes of sphingolipids in wild Cordyceps and its mycelia is sphingoid bases/ceramides > phosphosphingolipids > glycosphingolipids. However, remarkable sphingolipid differences between wild Cordyceps and its mycelia were observed. One is that sphingoid base is the dominant sphingolipid in wild Cordyceps, whereas ceramide is the major sphingolipid in mycelia. Another difference is that the abundance of sphingomyelins in wild Cordyceps is almost 10-folds higher than those in most mycelia. The third one is that mycelia contain more inositol phosphorylceramides and glycosphingolipids than wild Cordyceps. Multivariate analysis was further employed to visualize the difference among wild Cordyceps and different mycelia, leading to the identification of respective sphingolipids as potential chemical markers for the differentiation of wild Cordyceps and its related mycelia. This study represents the first report on the quantitative profiling of sphingolipids in wild Cordyceps and its related mycelia, which provided comprehensive chemical evidence for the quality control and rational utilization of wild Cordyceps and its mycelia. PMID:26868933

  15. Sphingolipids and Plant Defense/Disease: The “Death” Connection and Beyond

    PubMed Central

    Berkey, Robert; Bendigeri, Dipti; Xiao, Shunyuan

    2012-01-01

    Sphingolipids comprise a major class of structural materials and lipid signaling molecules in all eukaryotic cells. Over the past two decades, there has been a phenomenal growth in the study of sphingolipids (i.e., sphingobiology) at an average rate of ∼1000 research articles per year. Sphingolipid studies in plants, though accounting for only a small fraction (∼6%) of the total number of publications, have also enjoyed proportionally rapid growth in the past decade. Concomitant with the growth of sphingobiology, there has also been tremendous progress in our understanding of the molecular mechanisms of plant innate immunity. In this review, we (i) cross examine and analyze the major findings that establish and strengthen the intimate connections between sphingolipid metabolism and plant programmed cell death (PCD) associated with plant defense or disease; (ii) highlight and compare key bioactive sphingolipids involved in the regulation of plant PCD and possibly defense; (iii) discuss the potential role of sphingolipids in polarized membrane/protein trafficking and formation of lipid rafts as subdomains of cell membranes in relation to plant defense; and (iv) where possible, attempt to identify potential parallels for immunity-related mechanisms involving sphingolipids across kingdoms. PMID:22639658

  16. Brave little yeast, please guide us to thebes: sphingolipid function in S. cerevisiae.

    PubMed

    Schneiter, R

    1999-12-01

    Sphingolipids typically cover the exoplasmic leaflet of the plasma membrane of eukaryotic cells. They differ from the more abundant glycerophospholipids in that they contain ceramide instead of diacylglycerol as a hydrophobic anchor. Why did nature choose to invent this complex class of lipids, and why do eukaryotic cells follow elaborate remodelling pathways in order to generate dozens to hundreds of different molecular species of sphingolipid, depending on cell type? Yeast may, once again, serve as a model to dissect sphingolipid function at various levels. Almost the complete pathway for sphingolipid synthesis in yeast has been uncovered during the past two decades. More recently, key enzymes in sphingolipid degradation and signalling have been identified. Together with a wealth of genetic data obtained from the characterization of various suppressor mutants, this information now allows for an unprecedented analysis of sphingolipid function in this organism. This overview summarizes recent data on sphingolipid function in cell signalling, their role in the heat-stress response and Ca(2+) homeostasis, and addresses their function in transport of glycosylphosphatidylinositol-anchored proteins.

  17. Gene expression of sphingolipid metabolism pathways is altered in hidradenitis suppurativa.

    PubMed

    Dany, Mohammed; Elston, Dirk

    2017-08-01

    Hidradenitis suppurativa (HS) is a debilitating skin disease characterized by painful recurrent nodules and abscesses caused by chronic inflammation. Early events in the development of HS are believed to occur in the folliculopilosebaceous unit; however, the signaling pathways behind this mechanism are unknown. Sphingolipids, such as ceramide, are essential components of the skin and appendages and have important structural and signaling roles. We sought to explore whether the gene expression of enzymes involved in sphingolipid metabolic pathways is altered in HS. A microarray data set including 30 samples was used to compare the expression of sphingolipid-related enzymes in inflammatory skin lesions from HS patients (n = 17) with the expression in clinically healthy skin tissue (n = 13). Differential expression of sphingolipid metabolism-related genes was analyzed using Gene Expression Omnibus 2R. HS lesional skin samples have significantly decreased expression of enzymes generating ceramide and sphingomyelin, increased expression of enzymes catabolizing ceramide to sphingosine, and increased expression of enzymes converting ceramide to galactosylceramide and gangliosides. Limitations of this study include assessing the expression of sphingolipid-related enzymes without assessing the levels of the related sphingolipids. Our study suggests that sphingolipid metabolism is altered in HS lesional skin compared with normal skin. Copyright © 2017 American Academy of Dermatology, Inc. Published by Elsevier Inc. All rights reserved.

  18. Converging roles for sphingolipids and cell stress in the progression of neurological dysfunction in AIDS

    PubMed Central

    Haughey, Norman J.; Steiner, Joesph; Nath, Avindra; McArthur, Justin; Sacktor, Ned; Pardo, Carlos; Bandaru, Veera Venkata Ratnam

    2009-01-01

    Sphingolipids are a class of lipids enriched in the central nervous system that have important roles in signal transduction. Recent advances in our understanding of how sphingolipids are involved in the control of life and death signaling have uncovered roles for these lipids in the neuropathogenesis of HIV-associated neurocognitive disorders (HAND). In this review we briefly summarize the molecular mechanisms involved in the pathological production of the toxic sphingolipid, ceramide and address questions of how cytokine and cellular stress pathways that are perturbed in HAND converge to deregulate ceramide-associated signaling. PMID:18508574

  19. Tackling the biophysical properties of sphingolipids to decipher their biological roles.

    PubMed

    Carreira, Ana C; Ventura, Ana E; Varela, Ana R P; Silva, Liana C

    2015-06-01

    From the most simple sphingoid bases to their complex glycosylated derivatives, several sphingolipid species were shown to have a role in fundamental cellular events and/or disease. Increasing evidence places lipid-lipid interactions and membrane structural alterations as central mechanisms underlying the action of these lipids. Understanding how these molecules exert their biological roles by studying their impact in the physical properties and organization of membranes is currently one of the main challenges in sphingolipid research. Herein, we review the progress in the state-of-the-art on the biophysical properties of sphingolipid-containing membranes, focusing on sphingosine, ceramides, and glycosphingolipids.

  20. An introduction to sphingolipid metabolism and analysis by new technologies.

    PubMed

    Chen, Yanfeng; Liu, Ying; Sullards, M Cameron; Merrill, Alfred H

    2010-12-01

    Sphingolipids (SP) are a complex class of molecules found in essentially all eukaryotes and some prokaryotes and viruses where they influence membrane structure, intracellular signaling, and interactions with the extracellular environment. Because of the combinatorial nature of their biosynthesis, there are thousands of SP subspecies varying in the lipid backbones and complex phospho- and glycoheadgroups. Therefore, comprehensive or "sphingolipidomic" analyses (structure-specific, quantitative analyses of all SP, or at least all members of a critical subset) are needed to know which and how much of these subspecies are present in a system as a step toward understanding their functions. Mass spectrometry and related novel techniques are able to quantify a small fraction, but nonetheless a substantial number, of SP and are beginning to provide information about their localization. This review summarizes the basic metabolism of SP and state-of-art mass spectrometric techniques that are producing insights into SP structure, metabolism, functions, and some of the dysfunctions of relevance to neuromedicine.

  1. Sphingolipids from the human fungal pathogen Aspergillus fumigatus.

    PubMed

    Fontaine, Thierry

    2017-10-01

    Sphingolipids (SPLs) are key components of the plasma membrane in yeast and filamentous fungi. These molecules are involved in a number of cellular processes, and particularly, SGLs are essential components of the highly polarized fungal growth where they are required for the formation of the polarisome organization at the hyphal apex. Aspergillus fumigatus, a human fungal pathogen, produce SGLs that are discriminated into neutral cerebrosides, glycosylinositolphosphoceramides (GIPCs) and glycosylphosphatidylinositol (GPI) anchors. In addition to complex hydrophilic head groups of GIPCs, A. fumigatus is, to date, the sole fungus that produces a GPI-anchored polysaccharide. These SPLs follow three different biosynthetic pathways. Genetics blockage leading to the inhibition of any SPL biosynthesis or to the alteration of the structure of SPL induces growth and virulence defects. The complete lipid moiety of SPLs is essential for the lipid microdomain organization and their biosynthetic pathways are potential antifungal targets but remains understudied. Copyright © 2017. Published by Elsevier B.V.

  2. Sphingolipid trafficking and purification in Chlamydia trachomatis-infected cells

    PubMed Central

    2012-01-01

    Chlamydia trachomatis is an obligate intracellular human pathogen, which lacks a system that allows genetic manipulation. Therefore, chlamydial researchers must manipulate the host cell to better understand chlamydial biology. Host-derived lipid acquisition is critical for chlamydial survival within the host. Hence, the ability to track and purify sphingolipids in/from chlamydial infected cells has become an integral part of pivotal studies in chlamydial biology. This Unit outlines protocols that provide details about labeling eukaryotic cells with exogenous lipids to examine Golgi-derived lipid trafficking to the chlamydial inclusion and then performing imaging studies or lipid extractions for quantification. Details are provided to allow these protocols to be applied to subconfluent, polarized or siRNA knockdown cells. In addition, one will find important experimental design considerations and techniques. These methods are powerful tools to aid in the understanding of mechanisms which allow C. trachomatis to manipulate and usurp host cell trafficking pathways. PMID:23184593

  3. Plasma sphingolipids in HIV-associated chronic obstructive pulmonary disease

    PubMed Central

    Hodgson, Shane; Griffin, Timothy J; Reilly, Cavan; Harvey, Stephen; Witthuhn, Bruce A; Sandri, Brian J; Wendt, Chris H

    2017-01-01

    Introduction Chronic obstructive pulmonary disease (COPD) is a significant cause of morbidity in persons living with HIV (PLWH) and HIV appears to uniquely cause COPD, independent of smoking. The mechanisms by which HIV leads to COPD are not clear. The objective of this study was to identify metabolomic biomarkers and potential mechanistic pathways of HIV-associated COPD (HIV-COPD). Methods We performed case–control metabolite profiling via mass spectrometry in plasma from 38 individuals with HIV-COPD (cases), comparing to matched controls with/without HIV and with/without COPD. Untargeted metabolites of interest were identified with liquid chromatography with mass spectrometry (LC-MS/mass spectrometry (MS)), and targeted metabolomics for tryptophan (Trp) and kynurenine (Kyn) were measured by selective reaction monitoring (SRM) with LC-MS/MS. We used mixed-effects models to compare metabolite concentrations in cases compared with controls while controlling for relevant biological variables. Results We identified 1689 analytes associated with HIV-COPD at a false discovery rate (FDR) of 10%. In PLWH, we identified 263 analytes (10% FDR) between those with and without COPD. LC MS/MS identified Trp and 17 lipids, including sphingolipids and diacylglycerol. After adjusting for relevant covariates, the Kyn/Trp ratio measured by SRM was significantly higher in PLWH (p=0.022), but was not associated with COPD status (p=0.95). Conclusions There is a unique metabolite profile in HIV-COPD that includes sphingolipids. Trp metabolism is increased in HIV, but does not appear to independently contribute to HIV-COPD. Trial registration numbers NCT01810289, NCT01797367, NCT00608764. PMID:28409005

  4. Specific sphingolipid content decrease in Cerkl knockdown mouse retinas

    PubMed Central

    Garanto, Alejandro; Mandal, Nawajes A.; Egido-Gabás, Meritxell; Marfany, Gemma; Fabriàs, Gemma; Anderson, Robert E.; Casas, Josefina; Gonzàlez-Duarte, Roser

    2014-01-01

    Sphingolipids (SPLs) are finely tuned structural compounds and bioactive molecules involved in membrane fluidity and cellular homeostasis. The core sphingolipid, ceramide (CER), and its derivatives, regulate several crucial processes in neuronal cells, among them cell differentiation, cell–cell interactions, membrane conductance, synaptic transmission, and apoptosis. Mutations in Ceramide Kinase-Like (CERKL) cause autosomal recessive Retinitis Pigmentosa and Cone Rod Dystrophy. The presence of a conserved lipid kinase domain and the overall similarity with CERK suggested that CERKL might play a role in the SPL metabolism as a CER kinase. Unfortunately, CERKL function and substrate(s), as well as its contribution to the retinal etiopathology, remain as yet unknown. In this work we aimed to characterize the mouse retinal sphingolipidome by UPLC-TOF to first, thoroughly investigate the SPL composition of the murine retina, compare it to our Cerkl −/− model, and finally assess new possible CERKL substrates by phosphorus quantification and protein-lipid overlay. Our results showed a consistent and notable decrease of the retinal SPL content (mainly ranging from 30% to 60%) in the Cerkl −/− compared to WT retinas, which was particularly evident in the glucosyl/galactosyl ceramide species (Glc/GalCer) whereas the phospholipids and neutral lipids remained unaltered. Moreover, evidence in favor of CERKL binding to GlcCer, GalCer and sphingomyelin has been gathered. Altogether, these results highlight the involvement of CERKL in the SPL metabolism, question its role as a kinase, and open new scenarios concerning its function. PMID:23501591

  5. Sphingolipids, Lipid Rafts, and Giardial Encystation: The Show Must Go On.

    PubMed

    Mendez, Tavis L; De Chatterjee, Atasi; Duarte, Trevor; De Leon, Joaquin; Robles-Martinez, Leobarda; Das, Siddhartha

    2015-09-01

    Sphingolipids are sphingosine-based phospholipids, which are present in the plasma and endomembranes of many eukaryotic cells. These lipids are involved in various cellular functions, including cell growth, differentiation, and apoptosis. In addition, sphingolipid and cholesterol-enriched membrane microdomains (also called "lipid rafts") contain a set of proteins and lipids, which take part in the signaling process in response to intra- or extracellular stimuli. Recent findings suggest that sphingolipids, especially glucosylceramide, play a critical role in inducing encystation and maintaining the cyst viability in Giardia. Similarly, the assembly/disassembly of lipid rafts modulates the encystation and cyst production of this ubiquitous enteric parasite. In this review article, we discuss the overall progress in the field and examine whether sphingolipids and lipid rafts can be used as novel targets for designing therapies to control infection by Giardia, which is rampant in developing countries, where children are especially vulnerable.

  6. Trafficking and Functions of Bioactive Sphingolipids: Lessons from Cells and Model Membranes

    PubMed Central

    Zhou, Kecheng; Blom, Tomas

    2015-01-01

    Ceramide and sphingosine and their phosphorylated counterparts are recognized as “bioactive sphingolipids” and modulate membrane integrity, the activity of enzymes, or act as ligands of G protein-coupled receptors. The subcellular distribution of the bioactive sphingolipids is central to their function as the same lipid can mediate diametrically opposite effects depending on its location. To ensure that these lipids are present in the right amount and in the appropriate organelles, cells employ selective lipid transport and compartmentalize sphingolipid-metabolizing enzymes to characteristic subcellular sites. Our knowledge of key mechanisms involved in sphingolipid signaling and trafficking has increased substantially in the past decades—thanks to advances in biochemical and cell biological methods. In this review, we focus on the bioactive sphingolipids and discuss how the combination of studies in cells and in model membranes have contributed to our understanding of how they behave and function in living organisms. PMID:26715852

  7. Sphingolipids Are Dual Specific Drug Targets for the Management of Pulmonary Infections: Perspective

    PubMed Central

    Sharma, Lalita; Prakash, Hridayesh

    2017-01-01

    Sphingolipids are the major constituent of the mucus secreted by the cells of epithelial linings of lungs where they maintain the barrier functions and prevent microbial invasion. Sphingolipids are interconvertible, and their primary and secondary metabolites have both structural and functional roles. Out of several sphingolipid metabolites, sphingosine-1 phosphate (S1P) and ceramide are central molecules and decisive for sphingolipid signaling. These are produced by enzymatic activity of sphingosine kinase-1 (SK-1) upon the challenge with either biological or physiological stresses. S1P and ceramide rheostat are important for the progression of various pathologies, which are manifested by inflammatory cascade. S1P is a well-established secondary messenger and associated with various neuronal, metabolic, and inflammatory diseases other than respiratory infections such as Chlamydia pneumoniae, Streptococcus pneumoniae, and Mycobacterium tuberculosis. These pathogens are known to exploit sphingolipid metabolism for their opportunistic survival. Decreased sphingosine kinase activity/S1P content in the lung and peripheral blood of tuberculosis patients clearly indicated a dysregulation of sphingolipid metabolism during infection and suggest that sphingolipid metabolism is important for management of infection by the host. Our previous study has demonstrated that gain of SK-1 activity is important for the maturation of phagolysosomal compartment, innate activation of macrophages, and subsequent control of mycobacterial replication/growth in macrophages. Furthermore, S1P-mediated amelioration of lung pathology and disease severity in TB patients is believed to be mediated by the selective activation or rearrangement of various S1P receptors (S1PR) particularly S1PR2, which has been effective in controlling respiratory fungal pathogens. Therefore, such specificity of S1P–S1PR would be paramount for triggering inflammatory events, subsequent activation, and fostering

  8. Symbiont-derived sphingolipids modulate mucosal homeostasis and B cells in teleost fish

    PubMed Central

    Sepahi, Ali; Cordero, Héctor; Goldfine, Howard; Esteban, Maria Ángeles; Salinas, Irene

    2016-01-01

    Symbiotic bacteria and mucosal immunoglobulins have co-evolved for millions of years in vertebrate animals. Symbiotic bacteria products are known to modulate different aspects of the host immune system. We recently reported that Flectobacillus major is a predominant species that lives in the gill and skin mucosal surfaces of rainbow trout (Oncorhynchus mykiss). F. major is known to produce sphingolipids of a unique molecular structure. Here we propose a role for F. major and its sphingolipids in the regulation of B cell populations in rainbow trout, as well as an essential role for sphingolipids in trout mucosal homeostasis. We found that F. major-specific IgT titers are confined to the gill and skin mucus, whereas F. major-specific IgM titers are only detected in serum. Live F. major cells are able to stimulate sustained IgT expression and secretion in gills. F. major sphingolipids modulate the growth of trout total skin and gill symbiotic bacteria. In vivo systemic administration of F. major sphingolipids changes the proportion of IgT+ to IgM+ B cells in trout HK. These results demonstrate the key role of the symbiont F. major and its sphingolipids in mucosal homeostasis via the modulation of mucosal and systemic Igs and B cells. PMID:27966609

  9. Symbiont-derived sphingolipids modulate mucosal homeostasis and B cells in teleost fish.

    PubMed

    Sepahi, Ali; Cordero, Héctor; Goldfine, Howard; Esteban, Maria Ángeles; Salinas, Irene

    2016-12-14

    Symbiotic bacteria and mucosal immunoglobulins have co-evolved for millions of years in vertebrate animals. Symbiotic bacteria products are known to modulate different aspects of the host immune system. We recently reported that Flectobacillus major is a predominant species that lives in the gill and skin mucosal surfaces of rainbow trout (Oncorhynchus mykiss). F. major is known to produce sphingolipids of a unique molecular structure. Here we propose a role for F. major and its sphingolipids in the regulation of B cell populations in rainbow trout, as well as an essential role for sphingolipids in trout mucosal homeostasis. We found that F. major-specific IgT titers are confined to the gill and skin mucus, whereas F. major-specific IgM titers are only detected in serum. Live F. major cells are able to stimulate sustained IgT expression and secretion in gills. F. major sphingolipids modulate the growth of trout total skin and gill symbiotic bacteria. In vivo systemic administration of F. major sphingolipids changes the proportion of IgT(+) to IgM(+) B cells in trout HK. These results demonstrate the key role of the symbiont F. major and its sphingolipids in mucosal homeostasis via the modulation of mucosal and systemic Igs and B cells.

  10. Exploring the role of sphingolipid machinery during the epithelial to mesenchymal transition program using an integrative approach

    PubMed Central

    Meshcheryakova, Anastasia; Köfeler, Harald C.; Triebl, Alexander; Mungenast, Felicitas; Heinze, Georg; Gerner, Christopher; Zimmermann, Philip; Jaritz, Markus; Mechtcheriakova, Diana

    2016-01-01

    The epithelial to mesenchymal transition (EMT) program is activated in epithelial cancer cells and facilitates their ability to metastasize based on enhanced migratory, proliferative, anti-apoptotic, and pluripotent capacities. Given the fundamental impact of sphingolipid machinery to each individual process, the sphingolipid-related mechanisms might be considered among the most prominent drivers/players of EMT; yet, there is still limited knowledge. Given the complexity of the interconnected sphingolipid system, which includes distinct sphingolipid mediators, their synthesizing enzymes, receptors and transporters, we herein apply an integrative approach for assessment of the sphingolipid-associated mechanisms underlying EMT program. We created the sphingolipid-/EMT-relevant 41-gene/23-gene signatures which were applied to denote transcriptional events in a lung cancer cell-based EMT model. Based on defined 35-gene sphingolipid/EMT-attributed signature of regulated genes, we show close associations between EMT markers, genes comprising the sphingolipid network at multiple levels and encoding sphingosine 1-phosphate (S1P)-/ceramide-metabolizing enzymes, S1P and lysophosphatidic acid (LPA) receptors and S1P transporters, pluripotency genes and inflammation-related molecules, and demonstrate the underlying biological pathways and regulators. Mass spectrometry-based sphingolipid analysis revealed an EMT-attributed shift towards increased S1P and LPA accompanied by reduced ceramide levels. Notably, using transcriptomics data across various cell-based perturbations and neoplastic tissues (24193 arrays), we identified the sphingolipid/EMT signature primarily in lung adenocarcinoma tissues; besides, bladder, colorectal and prostate cancers were among the top-ranked. The findings also highlight novel regulatory associations between influenza virus and the sphingolipid/EMT-associated mechanisms. In sum, data propose the multidimensional contribution of sphingolipid machinery

  11. New lipoxygenase and cholinesterase inhibitory sphingolipids from Carthamus oxyacantha.

    PubMed

    Dilshad, Muhammad; Riaz, Naheed; Saleem, Muhammad; Shafiq, Nusrat; Ashraf, Muhammad; Ismail, Tayaba; Rafiq, Hafiza Mehwish; Jabbar, Abdul

    2016-08-01

    Two new sphingolipids: oxyacanthin A [(2S,3S,4R)-2-{[(2R,5E)-2-hydroxyoctadec-5-enoyl]amino}hexaeicosane-1,3,4-triol; 1] and B [(2S,3S,4R)-2-{[(2R,5E)-2-hydroxyoctadec-5-enoyl]amino}hexaeicosane-1,3,4-triol-1-O-β-D-glucopyranoside; 2], together with 1-octacosanol, β-sitosterol, β-sitosterol 3-O-β-D-glucopyranoside and luteolin 7-O-β-glucopyranoside were isolated from the methanolic extract of the whole plant of Carthamus oxyacantha. Their structures were elucidated using (1)H and (13)C NMR spectra and 2D NMR analyses (HMQC, HMBC and COSY) in combination with mass spectrometry (EI-MS, HR-EI-MS, FAB-MS and HR-FAB-MS) experiments and in comparison with the literature data of the related compounds. Both the compounds 1 and 2 showed inhibitory potential against lipoxygenase (LOX) in a concentration-dependent manner with IC50 values 83.3 ± 1.3 and 245.7 ± 1.1 µM, whereas compound 2 showed inhibition against enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) with IC50 values 65.3 ± 0.1 and 93.6 ± 0.1 µM, respectively.

  12. An Introduction to Sphingolipid Metabolism and Analysis by New Technologies

    PubMed Central

    Chen, Yanfeng; Liu, Ying; Sullards, M. Cameron

    2010-01-01

    Sphingolipids (SP) are a complex class of molecules found in essentially all eukaryotes and some prokaryotes and viruses where they influence membrane structure, intracellular signaling, and interactions with the extracellular environment. Because of the combinatorial nature of their biosynthesis, there are thousands of SP subspecies varying in the lipid backbones and complex phospho- and glycoheadgroups. Therefore, comprehensive or “sphingolipidomic” analyses (structure-specific, quantitative analyses of all SP, or at least all members of a critical subset) are needed to know which and how much of these subspecies are present in a system as a step toward understanding their functions. Mass spectrometry and related novel techniques are able to quantify a small fraction, but nonetheless a substantial number, of SP and are beginning to provide information about their localization. This review summarizes the basic metabolism of SP and state-of-art mass spectrometric techniques that are producing insights into SP structure, metabolism, functions, and some of the dysfunctions of relevance to neuromedicine. PMID:20680704

  13. Stored platelets alter glycerophospholipid and sphingolipid species, which are differentially transferred to newly released extracellular vesicles.

    PubMed

    Pienimaeki-Roemer, Annika; Ruebsaamen, Katharina; Boettcher, Alfred; Orsó, Evelyn; Scherer, Max; Liebisch, Gerhard; Kilalic, Dzenan; Ahrens, Norbert; Schmitz, Gerd

    2013-03-01

    Stored platelet concentrates (PLCs) for transfusion develop a platelet storage lesion (PSL), resulting in decreased platelet (PLT) viability and function. The processes leading to PSL have not been described in detail and no data describe molecular changes occurring in all three components of stored PLCs: PLTs, PLC extracellular vesicles (PLC-EVs), and plasma. Fifty PLCs from healthy individuals were stored under standard blood banking conditions for 5 days. Changes in cholesterol, glycerophospholipid, and sphingolipid species were analyzed in PLTs, PLC-EVs, and plasma by mass spectrometry and metabolic labeling. Immunoblots were performed to compare PLT and PLC-EV protein expression. During 5 days, PLTs transferred glycerophospholipids, cholesterol, and sphingolipids to newly formed PLC-EVs, which increased corresponding lipids by 30%. Stored PLTs significantly increased ceramide (Cer; +53%) and decreased sphingosine-1-phosphate (-53%), shifting sphingolipid metabolism toward Cer. In contrast, plasma accumulated minor sphingolipids. Compared to PLTs, fresh PLC-EVs were enriched in lysophosphatidic acid (60-fold) and during storage showed significant increases in cholesterol, sphingomyelin, dihydrosphingomyelin, plasmalogen, and lysophosphatidylcholine species, as well as accumulation of apolipoproteins A-I, E, and J/clusterin. This is the first detailed analysis of lipid species in all PLC components during PLC storage, which might reflect mechanisms active during in vivo PLT senescence. Stored PLTs reduce minor sphingolipids and shift sphingolipid metabolism toward Cer, whereas in the plasma fraction minor sphingolipids increase. The composition of PLC-EVs resembles that of lipid rafts and confirms their role as carriers of bioactive molecules and master regulators in vascular disease. © 2012 American Association of Blood Banks.

  14. Temporal changes in sphingolipids and systemic insulin sensitivity during the transition from gestation to lactation

    PubMed Central

    Rico, J. Eduardo; Saed Samii, Sina; Mathews, Alice T.; Lovett, Jacqueline; Haughey, Norman J.; McFadden, Joseph W.

    2017-01-01

    Reduced insulin action develops naturally during the peripartum to ensure maternal nutrient delivery to the fetus and neonate. However, increased insulin resistance can facilitate excessive lipolysis which in turn promotes metabolic disease in overweight dairy cattle. Increased fatty acid availability favors the accumulation of the sphingolipid ceramide and is implicated in the pathogenesis of insulin resistance, however, the relationship between sphingolipid metabolism and insulin resistance during the peripartum remains largely unknown. Our objectives were to characterize temporal responses in plasma and tissue sphingolipids in lean and overweight peripartal cows and to establish the relationships between sphingolipid supply and lipolysis, hepatic lipid deposition, and systemic insulin action. Twenty-one multiparous lean and overweight Holstein cows were enrolled in a longitudinal study spanning the transition from gestation to lactation (d -21 to 21, relative to parturition). Plasma, liver, and skeletal muscle samples were obtained, and sphingolipids were profiled using LC/MS/MS. Insulin sensitivity was assessed utilizing intravenous insulin and glucose challenges. Our results demonstrated the following: first, insulin resistance develops postpartum concurrently with increased lipolysis and hepatic lipid accumulation; second, ceramides and glycosylated ceramides accumulate during the transition from gestation to lactation and are further elevated in overweight cows; third, ceramide accrual is associated with lipolysis and liver lipid accumulation, and C16:0- and C24:0-ceramide are inversely associated with systemic insulin sensitivity postpartum; fourth, plasma sphingomyelin, a potential source of ceramides reaches a nadir at parturition and is closely associated with feed intake; fifth, select sphingomyelins are lower in the plasma of overweight cows during the peripartal period. Our results demonstrate that dynamic changes occur in peripartal sphingolipids

  15. Characterization of sphingolipids from sunflower seeds with altered fatty acid composition.

    PubMed

    Salas, Joaquín J; Markham, Jonathan E; Martínez-Force, Enrique; Garcés, Rafael

    2011-12-14

    Sphingolipids are a group of lipids that are derived from long-chain 1,3-dihydroxy-2-amino bases and that are involved in important processes in plants. Long-chain bases are usually found bound to long-chain fatty acids forming ceramides, the lipophilic moiety of the most common sphingolipid classes found in plant tissues: glucosyl-ceramides and glucosyl inositol phosphoryl-ceramides (GIPCs). The developing sunflower seed kernel is a tissue rich in sphingolipids, although, importantly, its glycerolipid composition can vary if some steps of the fatty acid synthesis are altered. Here, the sphingolipid composition of the seed from different sunflower mutants with altered fatty acid compositions was studied. The long-chain base composition and content were analyzed, and it was found to be similar in all of the mutants studied. The sphingolipid species were also determined by mass spectrometry, and some differences were found in highly saturated sunflower mutants, which contained higher levels of GIPC, ceramides, and hydroxyl-ceramides.

  16. Stress-Activated Degradation of Sphingolipids Regulates Mitochondrial Function and Cell Death in Yeast

    PubMed Central

    Manzanares-Estreder, Sara; Pascual-Ahuir, Amparo

    2017-01-01

    Sphingolipids are regulators of mitochondria-mediated cell death in higher eukaryotes. Here, we investigate how changes in sphingolipid metabolism and downstream intermediates of sphingosine impinge on mitochondrial function. We found in yeast that within the sphingolipid degradation pathway, the production via Dpl1p and degradation via Hfd1p of hexadecenal are critical for mitochondrial function and cell death. Genetic interventions, which favor hexadecenal accumulation, diminish oxygen consumption rates and increase reactive oxygen species production and mitochondrial fragmentation and vice versa. The location of the hexadecenal-degrading enzyme Hfd1p in punctuate structures all along the mitochondrial network depends on a functional ERMES (endoplasmic reticulum-mitochondria encounter structure) complex, indicating that modulation of hexadecenal levels at specific ER-mitochondria contact sites might be an important trigger of cell death. This is further supported by the finding that externally added hexadecenal or the absence of Hfd1p enhances cell death caused by ectopic expression of the human Bax protein. Finally, the induction of the sphingolipid degradation pathway upon stress is controlled by the Hog1p MAP kinase. Therefore, the stress-regulated modulation of sphingolipid degradation might be a conserved way to induce cell death in eukaryotic organisms. PMID:28845213

  17. Establishment of HeLa Cell Mutants Deficient in Sphingolipid-Related Genes Using TALENs

    PubMed Central

    Yamaji, Toshiyuki; Hanada, Kentaro

    2014-01-01

    Sphingolipids are essential components in eukaryotes and have various cellular functions. Recent developments in genome-editing technologies have facilitated gene disruption in various organisms and cell lines. We here show the disruption of various sphingolipid metabolic genes in human cervical carcinoma HeLa cells by using transcription activator-like effector nucleases (TALENs). A TALEN pair targeting the human CERT gene (alternative name COL4A3BP) encoding a ceramide transport protein induced a loss-of-function phenotype in more than 60% of HeLa cells even though the cell line has a pseudo-triploid karyotype. We have isolated several loss-of-function mutant clones for CERT, UGCG (encoding glucosylceramide synthase), and B4GalT5 (encoding the major lactosylceramide synthase), and also a CERT/UGCG double-deficient clone. Characterization of these clones supported previous proposals that CERT primarily contributes to the synthesis of SM but not GlcCer, and that B4GalT5 is the major LacCer synthase. These newly established sphingolipid-deficient HeLa cell mutants together with our previously established stable transfectants provide a ‘sphingolipid-modified HeLa cell panel,’ which will be useful to elucidate the functions of various sphingolipid species against essentially the same genomic background. PMID:24498430

  18. Sphingolipids in Genetic and Acquired Forms of Chronic Kidney Diseases.

    PubMed

    Ueda, Norishi

    2017-01-12

    Sphingolipids (SLs) regulate apoptosis, proliferation, and stress response. SLs, including ceramide, glycosphingolipids (glucosylceramide, lactosylceramide, and gangliosides) and sphingosine-1-phosphate (S1P), play a role in the pathogenesis and progression of genetic (lysosomal storage disease, congenital nephrotic syndrome and polycystic kidney disease) and non-genetic forms of chronic kidney diseases (CKDs). SLs metabolism defects promote complications (cardiovascular events, etc.) via oxidant stress in CKDs. A balancing role of apoptotic SLs and anti-apoptotic S1P is crucial in the regulation of glomerular injury and complications associated with CKDs. Interaction between SLs, endothelial function and renin-angiotensin-aldosterone system (RAAS) plays an important role in the regulation of glomerular injury. SLs affect mitochondrial function that regulate the opening of mitochondrial permeability transition (MPT) pore, mitochondrial outer membrane permeability (MOMP), generation of reactive oxygen species (ROS), Bcl-2 family proteins, leading to cytochrome c release and caspase activation, leading to apoptosis, and regulate glomerular cell proliferation or renal fibrosis. Interaction between SLs, endothelial function and RAAS plays a role in the regulation of glomerular injury. This review article summarizes the current evidence supporting a role of SLs metabolism defects in the pathogenesis and progression of glomerular injury and discusses a role of mitochondria, including MPT pore, MOMP, ROS, Bcl-2 family proteins, interaction between SLs, endothelial function and RAAS, and SLs-induced downstream signaling events in CKDs. Crosstalk between these factors plays a role in the pathogenesis and progression of CKDs. Therapeutic strategy of targeting SLs metabolism defects for CKDs through modulation of the enzymes responsible for SLs metabolism defects are also discussed.

  19. Newly identified essential amino acid residues affecting ^8-sphingolipid desaturase activity revealed by site-directed mutagenesis

    USDA-ARS?s Scientific Manuscript database

    In order to identify amino acid residues crucial for the enzymatic activity of ^8-sphingolipid desaturases, a sequence comparison was performed among ^8-sphingolipid desaturases and ^6-fatty acid desaturase from various plants. In addition to the known conserved cytb5 (cytochrome b5) HPGG motif and...

  20. Loss of Frataxin activates the iron/sphingolipid/PDK1/Mef2 pathway in mammals.

    PubMed

    Chen, Kuchuan; Ho, Tammy Szu-Yu; Lin, Guang; Tan, Kai Li; Rasband, Matthew N; Bellen, Hugo J

    2016-11-30

    Friedreich's ataxia (FRDA) is an autosomal recessive neurodegenerative disease caused by mutations in Frataxin (FXN). Loss of FXN causes impaired mitochondrial function and iron homeostasis. An elevated production of reactive oxygen species (ROS) was previously proposed to contribute to the pathogenesis of FRDA. We recently showed that loss of frataxin homolog (fh), a Drosophila homolog of FXN, causes a ROS independent neurodegeneration in flies (Chen et al., 2016). In fh mutants, iron accumulation in the nervous system enhances the synthesis of sphingolipids, which in turn activates 3-phosphoinositide dependent protein kinase-1 (Pdk1) and myocyte enhancer factor-2 (Mef2) to trigger neurodegeneration of adult photoreceptors. Here, we show that loss of Fxn in the nervous system in mice also activates an iron/sphingolipid/PDK1/Mef2 pathway, indicating that the mechanism is evolutionarily conserved. Furthermore, sphingolipid levels and PDK1 activity are also increased in hearts of FRDA patients, suggesting that a similar pathway is affected in FRDA.

  1. The chemistry and biology of 6-hydroxyceramide, the youngest member of the human sphingolipid family.

    PubMed

    Kováčik, Andrej; Roh, Jaroslav; Vávrová, Kateřina

    2014-07-21

    Sphingolipids are crucial for the life of the cell. In land-dwelling mammals, they are equally important outside the cell-in the extracellular space of the skin barrier-because they prevent loss of water. Although a large body of research has elucidated many of the functions of sphingolipids, their extensive structural diversity remains intriguing. A new class of sphingolipids based on 6-hydroxylated sphingosine has recently been identified in human skin. Abnormal levels of these 6-hydroxylated ceramides have repeatedly been observed in atopic dermatitis; however, neither the biosynthesis nor the roles of these unique ceramide subclasses have been established in the human body. In this Minireview, we summarize the current knowledge of 6-hydroxyceramides, including their discovery, structure, stereochemistry, occurrence in healthy and diseased human epidermis, and synthetic approaches to 6-hydroxysphingosine and related ceramides.

  2. ORM Expression Alters Sphingolipid Homeostasis and Differentially Affects Ceramide Synthase Activity1[OPEN

    PubMed Central

    Kimberlin, Athen N.; Chen, Ming; Dunn, Teresa M.

    2016-01-01

    Sphingolipid synthesis is tightly regulated in eukaryotes. This regulation in plants ensures sufficient sphingolipids to support growth while limiting the accumulation of sphingolipid metabolites that induce programmed cell death. Serine palmitoyltransferase (SPT) catalyzes the first step in sphingolipid biosynthesis and is considered the primary sphingolipid homeostatic regulatory point. In this report, Arabidopsis (Arabidopsis thaliana) putative SPT regulatory proteins, orosomucoid-like proteins AtORM1 and AtORM2, were found to interact physically with Arabidopsis SPT and to suppress SPT activity when coexpressed with Arabidopsis SPT subunits long-chain base1 (LCB1) and LCB2 and the small subunit of SPT in a yeast (Saccharomyces cerevisiae) SPT-deficient mutant. Consistent with a role in SPT suppression, AtORM1 and AtORM2 overexpression lines displayed increased resistance to the programmed cell death-inducing mycotoxin fumonisin B1, with an accompanying reduced accumulation of LCBs and C16 fatty acid-containing ceramides relative to wild-type plants. Conversely, RNA interference (RNAi) suppression lines of AtORM1 and AtORM2 displayed increased sensitivity to fumonisin B1 and an accompanying strong increase in LCBs and C16 fatty acid-containing ceramides relative to wild-type plants. Overexpression lines also were found to have reduced activity of the class I ceramide synthase that uses C16 fatty acid acyl-coenzyme A and dihydroxy LCB substrates but increased activity of class II ceramide synthases that use very-long-chain fatty acyl-coenzyme A and trihydroxy LCB substrates. RNAi suppression lines, in contrast, displayed increased class I ceramide synthase activity but reduced class II ceramide synthase activity. These findings indicate that ORM mediation of SPT activity differentially regulates functionally distinct ceramide synthase activities as part of a broader sphingolipid homeostatic regulatory network. PMID:27506241

  3. Synthesis of Sphingolipids Impacts Survival of Porphyromonas gingivalis and the Presentation of Surface Polysaccharides

    PubMed Central

    Moye, Zachary D.; Valiuskyte, Kornelija; Dewhirst, Floyd E.; Nichols, Frank C.; Davey, Mary E.

    2016-01-01

    Bacteria alter the biophysical properties of their membrane lipids in response to environmental cues, such as shifts in pH or temperature. In essence, lipid composition determines membrane structure, which in turn influences many basic functions, such as transport, secretion, and signaling. Like other members of the phylum Bacteroidetes, the oral anaerobe Porphyromonas gingivalis possesses the ability to synthesize a variety of novel membrane lipids, including species of dihydroceramides that are distinct, yet similar in structure to sphingolipids produced by the human host. The role of dihydroceramides in the physiology and pathogenic potential of the human microbiota is only beginning to be explored; yet there is increasing data indicating that these lipids play a role in human diseases, such as periodontitis and multiple sclerosis. Here, we report on the identification of a gene (PG1780) in the chromosome of P. gingivalis strain W83 encoding a putative serine palmitoyltransferase, the enzyme that catalyzes the first step in sphingolipid biosynthesis. While we were able to detect dihydroceramides in whole lipid extracts of P. gingivalis cells as well as crude preparations of outer membrane vesicles, sphingolipids were absent in the PG1780 mutant strain. Moreover, we show that the synthesis of sphingolipids plays an essential role in the long-term survival of the organism as well as its resistance to oxidative stress. Further, a PG1780 mutant displayed much lower activity of cell-associated arginine and lysine gingipains, yet slightly higher activity in the corresponding culture supernates, which we hypothesize is due to altered membrane properties and anchoring of these proteases to the cell surface. In addition, we determined that sphingolipid production is critical to the presentation of surface polysaccharides, with the mutant strain displaying less K-antigen capsule and more anionic polysaccharide (APS). Overall, we have discovered that, in addition to their

  4. Tools for the analysis of metabolic flux through the sphingolipid pathway.

    PubMed

    Martínez-Montañés, Fernando; Schneiter, Roger

    2016-11-01

    Discerning the complex regulation of the enzymatic steps necessary for sphingolipid biosynthesis is facilitated by the utilization of tracers that allow a time-resolved analysis of the pathway dynamics without affecting the metabolic flux. Different strategies have been used and new tools are continuously being developed to probe the various enzymatic conversions that occur within this complex pathway. Here, we provide a short overview of the divergent fungal and mammalian sphingolipid biosynthetic routes, and of the tracers and methods that are frequently employed to follow the flux of intermediates throughout these pathways.

  5. Effect of sterol carrier protein-2 expression on sphingolipid distribution in plasma membrane lipid rafts/caveolae.

    PubMed

    Atshaves, Barbara P; Jefferson, John R; McIntosh, Avery L; Gallegos, Adalberto; McCann, Bonnie M; Landrock, Kerstin K; Kier, Ann B; Schroeder, Friedhelm

    2007-10-01

    Although sphingolipids are highly important signaling molecules enriched in lipid rafts/caveolae, relatively little is known regarding factors such as sphingolipid binding proteins that may regulate the distribution of sphingolipids to lipid rafts/caveolae of living cells. Since early work demonstrated that sterol carrier protein-2 (SCP-2) enhanced glycosphingolipid transfer from membranes in vitro, the effect of SCP-2 expression on sphingolipid distribution to lipid rafts/caveolae in living cells was examined. Using a non-detergent affinity chromatography method to isolate lipid rafts/caveolae and non-rafts from purified L-cell plasma membranes, it was shown that lipid rafts/caveolae were highly enriched in multiple sphingolipid species including ceramides, acidic glycosphingolipids (ganglioside GM1); neutral glycosphingolipids (monohexosides, dihexosides, globosides), and sphingomyelin as compared to non-raft domains. SCP-2 overexpression further enriched the content of total sphingolipids and select sphingolipid species in the lipid rafts/caveolae domains. Analysis of fluorescence binding and displacement data revealed that purified human recombinant SCP-2 exhibited high binding affinity (nanomolar range) for all sphingolipid classes tested. The binding affinity decreased in the following order: ceramides > acidic glycosphingolipid (ganglioside GM1) > neutral glycosphingolipid (monohexosides, hexosides, globosides) > sphingomyelin. Enrichment of individual sphingolipid classes to lipid rafts/caveolae versus non-rafts in SCP-2 expressing plasma membranes followed closely with those classes most strongly bound to SCP-2 (ceramides, GM1 > the neutral glycosphingolipids (monohexosides, dihexosides, and globosides) > sphingomyelin). Taken together these data suggested that SCP-2 acts to selectively regulate sphingolipid distribution to lipid rafts/caveolae in living cells.

  6. Sphingolipids Mediate Differential Echinocandin Susceptibility in Candida albicans and Aspergillus nidulans

    PubMed Central

    Healey, Kelley R.; Challa, Krishna K.; Edlind, Thomas D.

    2015-01-01

    The cell wall synthesis-inhibiting echinocandins, including caspofungin and micafungin, play important roles in the treatment of candidiasis and aspergillosis. Previous studies revealed that, in the haploid yeast Candida glabrata, sphingolipid biosynthesis pathway mutations confer caspofungin reduced susceptibility (CRS) but micafungin increased susceptibility (MIS). Here, we describe one Candida albicans strain (of 10 tested) that similarly yields CRS-MIS mutants at relatively high frequency. Mutants demonstrated increased levels of long-chain bases (sphingolipid pathway intermediates) and, unique to this strain, loss of His104/Pro104 heterozygosity in the TSC13-encoded enoyl reductase. CRS-MIS was similarly observed in a C. albicans homozygous fen1Δ fen12Δ laboratory strain and in diverse wild-type strains following exogenous long-chain-base treatment. Analogous to these results, CRS-MIS was demonstrated in an Aspergillus nidulans basA mutant encoding defective sphingolipid C4-hydroxylase and in its wild-type parent exposed to long-chain bases. Sphingolipids likely modulate echinocandin interaction with their Fks membrane target in all susceptible fungi, with potential implications for optimizing therapy with existing antifungals and the development of novel agents. PMID:25824222

  7. Low serum sphingolipids in children with attention deficit-hyperactivity disorder

    PubMed Central

    Henríquez-Henríquez, Marcela P.; Solari, Sandra; Quiroga, Teresa; Kim, Benjamin I.; Deckelbaum, Richard J.; Worgall, Tilla S.

    2015-01-01

    Background: Attention deficit-hyperactivity disorder (ADHD) is the most prevalent neuropsychiatric condition in childhood. ADHD is a multifactorial trait with a strong genetic component. One neurodevelopmental hypothesis is that ADHD is associated with a lag in brain maturation. Sphingolipids are essential for brain development and neuronal functioning, but their role in ADHD pathogenesis is unexplored. We hypothesized that serum sphingolipid levels distinguish ADHD patients from unaffected subjects. Methods: We characterized serum sphingolipid profiles of ADHD patients and two control groups: non-affected relatives and non-affected subjects without a family history of ADHD. Sphingolipids were measured by LC-MS/MS in 77 participants (28 ADHD patients, 28 related controls, and 21 unrelated controls). ADHD diagnosis was based on the Diagnostic and Statistical Manual of Mental Disorders (DSM IV-TR). Diagnostic criteria were assessed by two independent observers. Groups were compared by parametrical statistics. Results: Serum sphingomyelins C16:0, C18:0, C18:1, C24:1, ceramide C24:0, and deoxy-ceramide C24:1 were significantly decreased in ADHD patients at 20–30% relative reductions. In our sample, decreased serum sphingomyelin levels distinguished ADHD patients with 79% sensitivity and 78% specificity. Conclusions: Our results showed lower levels of all major serum sphingomyelins in ADHD. These findings may reflect brain maturation and affect neuro-functional pathways characteristic for ADHD. PMID:26379487

  8. Membrane sphingolipids as essential molecular signals for Bacteroides survival in the intestine

    PubMed Central

    An, Dingding; Na, Chongzheng; Bielawski, Jacek; Hannun, Yusuf A.; Kasper, Dennis L.

    2011-01-01

    As predominant intestinal symbiotic bacteria, Bacteroides are essential in maintaining the health of the normal mammalian host; in return, the host provides a niche with plentiful nutrients for the symbionts. However, the intestinal environment is replete with chemical, physical, and biological challenges that require mechanisms for prompt and adept sensing of and responses to stress if the bacteria are to survive. Herein we propose that to persist in the intestine Bacteroides take advantage of their unusual bacterial sphingolipids to mediate signaling pathways previously known to be available only to higher organisms. Sphingolipids convey diverse signal transduction and stress response pathways and have profound physiological impacts demonstrated in a variety of eukaryotic cell types. We propose a mechanism by which the formation of specific sphingolipid membrane microdomains initiates signaling cascades that facilitate survival strategies within the bacteria. Our preliminary data suggest that sphingolipid signaling plays an important role in Bacteroides physiology, enabling these bacteria to persist in the intestine and to perform other functions related to symbiosis. PMID:20855611

  9. Nogo-B regulates endothelial sphingolipid homeostasis to control vascular function and blood pressure

    PubMed Central

    Kothiya, Milankumar; Galvani, Sylvain; Obinata, Hideru; Bucci, Mariarosaria; Giordano, Frank J; Jiang, Xian-Cheng; Hla, Timothy; Di Lorenzo, Annarita

    2015-01-01

    Endothelial dysfunction is a critical factor in many cardiovascular diseases, including hypertension. Although lipid signaling has been implicated in endothelial dysfunction and cardiovascular disease, specific molecular mechanisms are poorly understood. Here we report that Nogo-B, a membrane protein of the endoplasmic reticulum, regulates endothelial sphingolipid biosynthesis with direct effects on vascular function and blood pressure. Nogo-B inhibits serine palmitoyltransferase, the rate-limiting enzyme of the de novo sphingolipid biosynthetic pathway, thereby controlling production of endothelial sphingosine 1-phosphate and autocrine, G protein–coupled receptor–dependent signaling by this metabolite. Mice lacking Nogo-B either systemically or specifically in endothelial cells are hypotensive, resistant to angiotensin II–induced hypertension and have preserved endothelial function and nitric oxide release. In mice that lack Nogo-B, pharmacological inhibition of serine palmitoyltransferase with myriocin reinstates endothelial dysfunction and angiotensin II–induced hypertension. Our study identifies Nogo-B as a key inhibitor of local sphingolipid synthesis and shows that autocrine sphingolipid signaling within the endothelium is critical for vascular function and blood pressure homeostasis. PMID:26301690

  10. The cellular ceramide transport protein CERT promotes Chlamydia psittaci infection and controls bacterial sphingolipid uptake.

    PubMed

    Koch-Edelmann, Sophia; Banhart, Sebastian; Saied, Essa M; Rose, Laura; Aeberhard, Lukas; Laue, Michael; Doellinger, Joerg; Arenz, Christoph; Heuer, Dagmar

    2017-10-01

    Chlamydiaceae are bacterial pathogens that cause diverse diseases in humans and animals. Despite their broad host and tissue tropism, all Chlamydia species share an obligate intracellular cycle of development and have evolved sophisticated mechanisms to interact with their eukaryotic host cells. Here, we have analysed interactions of the zoonotic pathogen Chlamydia psittaci with a human epithelial cell line. We found that C. psittaci recruits the ceramide transport protein (CERT) to its inclusion. Chemical inhibition and CRISPR/Cas9-mediated knockout of CERT showed that CERT is a crucial factor for C. psittaci infections thereby affecting different stages of the infection including inclusion growth and infectious progeny formation. Interestingly, the uptake of fluorescently labelled sphingolipids in bacteria inside the inclusion was accelerated in CERT-knockout cells indicating that C. psittaci can exploit CERT-independent sphingolipid uptake pathways. Moreover, the CERT-specific inhibitor HPA-12 strongly diminished sphingolipid transport to inclusions of infected CERT-knockout cells, suggesting that other HPA-12-sensitive factors are involved in sphingolipid trafficking to C. psittaci. Further analysis is required to decipher these interactions and to understand their contributions to bacterial development, host range, tissue tropism, and disease outcome. © 2017 John Wiley & Sons Ltd.

  11. Ganglioside and related-sphingolipid profiles are altered in a cellular model of Alzheimer's disease.

    PubMed

    Noel, Anastasia; Ingrand, Sabrina; Barrier, Laurence

    2017-06-01

    Sphingolipid-related issues are increasingly discussed to contribute to the neuropathological process of Alzheimer's disease (AD). In this study, gangliosides and related-sphingolipids (ceramides, neutral glycosphingolipids and sphingomyelins) were analyzed in neuroglioma (H4) cells expressing the Swedish mutation of the human amyloid precursor protein (H4APPsw) and compared with those of wild-type control H4 cells. These cells were chosen since H4APPsw cells were previously reported to reproduce well some essential features of AD. We found that H4APPsw cells exhibited a striking elevation of the simplest ganglioside GM3, an abnormality that was consistently reported in AD patients and animal models of AD. Concomitantly, the levels of both lactosylceramide (the immediate metabolic precursor of GM3) and ganglioside GD1a increased, suggesting a deregulation in the biosynthesis of gangliosides in the H4APPsw cells. Moreover, while the total ceramide level remained unaltered in H4APPsw cells, a shift in ceramide composition from long chain - to very long chain fatty acid-ceramide species was recorded. Because sphingolipid alterations occurring in H4APPsw cells were similar to those observed in transgenic mice and in human brains, this cellular model might be useful to further explore the complex role of sphingolipids in AD pathogenesis. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  12. Skin Ceramide Alterations in First-Episode Schizophrenia Indicate Abnormal Sphingolipid Metabolism

    PubMed Central

    Smesny, Stefan; Schmelzer, Christian E. H.; Hinder, Anke; Köhler, Alexandra; Schneider, Christiane; Rudzok, Maria; Schmidt, Ulrike; Milleit, Berko; Milleit, Christine; Nenadic, Igor; Sauer, Heinrich; Neubert, Reinhard H. H.; Fluhr, Joachim W.

    2013-01-01

    There is considerable evidence for specific pathology of lipid metabolism in schizophrenia, affecting polyunsaturated fatty acids and in particular sphingolipids. These deficits are assumed to interfere with neuronal membrane functioning and the development and maintenance of myelin sheaths. Recent studies suggest that some of these lipid pathologies might also be detected in peripheral skin tests. In this study, we examined different skin lipids and their relation to schizophrenia. We assessed epidermal lipid profiles in 22 first-episode antipsychotic-naïve schizophrenia patients and 22 healthy controls matched for age and gender using a hexan/ethanol extraction technique and combined high-performance thin-layer chromatography/gas-chromatography. We found highly significant increase of ceramide AH and NH/AS classes in patients and decrease of EOS and NP ceramide classes. This is the first demonstration of specific peripheral sphingolipid alterations in schizophrenia. The results support recent models of systemic lipid pathology and in particular of specific sphingolipids, which are crucial in neuronal membrane integrity. Given recent findings showing amelioration of psychopathology using fatty acid supplementation, our findings also bear relevance for sphingolipids as potential biomarkers of the disease. PMID:22589371

  13. Skin ceramide alterations in first-episode schizophrenia indicate abnormal sphingolipid metabolism.

    PubMed

    Smesny, Stefan; Schmelzer, Christian E H; Hinder, Anke; Köhler, Alexandra; Schneider, Christiane; Rudzok, Maria; Schmidt, Ulrike; Milleit, Berko; Milleit, Christine; Nenadic, Igor; Sauer, Heinrich; Neubert, Reinhard H H; Fluhr, Joachim W

    2013-07-01

    There is considerable evidence for specific pathology of lipid metabolism in schizophrenia, affecting polyunsaturated fatty acids and in particular sphingolipids. These deficits are assumed to interfere with neuronal membrane functioning and the development and maintenance of myelin sheaths. Recent studies suggest that some of these lipid pathologies might also be detected in peripheral skin tests. In this study, we examined different skin lipids and their relation to schizophrenia. We assessed epidermal lipid profiles in 22 first-episode antipsychotic-naïve schizophrenia patients and 22 healthy controls matched for age and gender using a hexan/ethanol extraction technique and combined high-performance thin-layer chromatography/gas-chromatography. We found highly significant increase of ceramide AH and NH/AS classes in patients and decrease of EOS and NP ceramide classes. This is the first demonstration of specific peripheral sphingolipid alterations in schizophrenia. The results support recent models of systemic lipid pathology and in particular of specific sphingolipids, which are crucial in neuronal membrane integrity. Given recent findings showing amelioration of psychopathology using fatty acid supplementation, our findings also bear relevance for sphingolipids as potential biomarkers of the disease.

  14. De Novo Sphingolipid Synthesis Is Essential for Viability, but Not for Transport of Glycosylphosphatidylinositol-Anchored Proteins, in African Trypanosomes▿

    PubMed Central

    Sutterwala, Shaheen S.; Creswell, Caleb H.; Sanyal, Sumana; Menon, Anant K.; Bangs, James D.

    2007-01-01

    De novo sphingolipid synthesis is required for the exit of glycosylphosphatidylinositol (GPI)-anchored membrane proteins from the endoplasmic reticulum in yeast. Using a pharmacological approach, we test the generality of this phenomenon by analyzing the transport of GPI-anchored cargo in widely divergent eukaryotic systems represented by African trypanosomes and HeLa cells. Myriocin, which blocks the first step of sphingolipid synthesis (serine + palmitate → 3-ketodihydrosphingosine), inhibited the growth of cultured bloodstream parasites, and growth was rescued with exogenous 3-ketodihydrosphingosine. Myriocin also blocked metabolic incorporation of [3H]serine into base-resistant sphingolipids. Biochemical analyses indicate that the radiolabeled lipids are not sphingomyelin or inositol phosphorylceramide, suggesting that bloodstream trypanosomes synthesize novel sphingolipids. Inhibition of de novo sphingolipid synthesis with myriocin had no adverse effect on either general secretory trafficking or GPI-dependent trafficking in trypanosomes, and similar results were obtained with HeLa cells. A mild effect on endocytosis was seen for bloodstream trypanosomes after prolonged incubation with myriocin. These results indicate that de novo synthesis of sphingolipids is not a general requirement for secretory trafficking in eukaryotic cells. However, in contrast to the closely related kinetoplastid Leishmania major, de novo sphingolipid synthesis is essential for the viability of bloodstream-stage African trypanosomes. PMID:17220466

  15. Toxoplasma gondii salvages sphingolipids from the host Golgi through the rerouting of selected Rab vesicles to the parasitophorous vacuole

    PubMed Central

    Romano, Julia D.; Sonda, Sabrina; Bergbower, Emily; Smith, Maria Elisa; Coppens, Isabelle

    2013-01-01

    The obligate intracellular protozoan Toxoplasma gondii actively invades mammalian cells and, upon entry, forms its own membrane-bound compartment, named the parasitophorous vacuole (PV). Within the PV, the parasite replicates and scavenges nutrients, including lipids, from host organelles. Although T. gondii can synthesize sphingolipids de novo, it also scavenges these lipids from the host Golgi. How the parasite obtains sphingolipids from the Golgi remains unclear, as the PV avoids fusion with host organelles. In this study, we explore the host Golgi–PV interaction and evaluate the importance of host-derived sphingolipids for parasite growth. We demonstrate that the PV preferentially localizes near the host Golgi early during infection and remains closely associated with this organelle throughout infection. The parasite subverts the structure of the host Golgi, resulting in its fragmentation into numerous ministacks, which surround the PV, and hijacks host Golgi–derived vesicles within the PV. These vesicles, marked with Rab14, Rab30, or Rab43, colocalize with host-derived sphingolipids in the vacuolar space. Scavenged sphingolipids contribute to parasite replication since alterations in host sphingolipid metabolism are detrimental for the parasite's growth. Thus our results reveal that T. gondii relies on host-derived sphingolipids for its development and scavenges these lipids via Golgi-derived vesicles. PMID:23615442

  16. Toxoplasma gondii salvages sphingolipids from the host Golgi through the rerouting of selected Rab vesicles to the parasitophorous vacuole.

    PubMed

    Romano, Julia D; Sonda, Sabrina; Bergbower, Emily; Smith, Maria Elisa; Coppens, Isabelle

    2013-06-01

    The obligate intracellular protozoan Toxoplasma gondii actively invades mammalian cells and, upon entry, forms its own membrane-bound compartment, named the parasitophorous vacuole (PV). Within the PV, the parasite replicates and scavenges nutrients, including lipids, from host organelles. Although T. gondii can synthesize sphingolipids de novo, it also scavenges these lipids from the host Golgi. How the parasite obtains sphingolipids from the Golgi remains unclear, as the PV avoids fusion with host organelles. In this study, we explore the host Golgi-PV interaction and evaluate the importance of host-derived sphingolipids for parasite growth. We demonstrate that the PV preferentially localizes near the host Golgi early during infection and remains closely associated with this organelle throughout infection. The parasite subverts the structure of the host Golgi, resulting in its fragmentation into numerous ministacks, which surround the PV, and hijacks host Golgi-derived vesicles within the PV. These vesicles, marked with Rab14, Rab30, or Rab43, colocalize with host-derived sphingolipids in the vacuolar space. Scavenged sphingolipids contribute to parasite replication since alterations in host sphingolipid metabolism are detrimental for the parasite's growth. Thus our results reveal that T. gondii relies on host-derived sphingolipids for its development and scavenges these lipids via Golgi-derived vesicles.

  17. Assessment of crosstalks between the Snf1 kinase complex and sphingolipid metabolism in S. cerevisiae via systems biology approaches.

    PubMed

    Borklu Yucel, Esra; Ulgen, Kutlu O

    2013-11-01

    Sphingolipids are essential building blocks of the plasma membranes and are highly bioactive in the regulation of diverse cellular functions and pathological processes, a fact which renders the sphingolipid metabolism an important research area. In this study, a computational framework was recruited for the reconstruction of a functional interaction network for sphingolipid metabolism in Baker's yeast, SSN. Gene Ontology (GO) annotations were integrated with functional interaction data of the BIOGRID database and the reconstructed protein interaction network was subjected to topological and descriptive analyses. SSN was of a scale-free nature, following a power law model with γ=1.41. Prominent processes of SSN revealed that the reconstructed network encapsulated the involvement of sphingolipid metabolism in vital cellular processes such as energy homeostasis, cell growth and/or death and synthesis of building blocks. To investigate the potential of SSN for predicting signal transduction pathways regulating and/or being regulated by sphingolipid biosynthesis in yeast, a case study involving the S. cerevisiae counterpart of AMP-activated protein kinase, the Snf1 kinase complex, was conducted. The mutant strain lacking the catalytic α subunit, snf1Δ/snf1Δ, had elevated inositol phosphorylceramide and mannosyl-inositol phosphorylceramide levels, and decreased mannosyl-diinositol phosphorylceramide levels compared to the wild type strain, revealing that Snf1p has a regulatory role in the sphingolipid metabolism. Transcriptome data belonging to that strain available in the literature were mapped onto SSN and the correlated SSN was further investigated to evaluate the possible crosstalk machineries where sphingolipids and Snf1p function in coordination, in other words the crosstalk points between sphingolipid-mediated and Snf1 kinase signalling. The subsequent investigation of the discovered candidate crosstalk processes by performing sensitivity experiments imply a

  18. An apparent association between glycosylphosphatidylinositol-anchored proteins and a sphingolipid in Tetrahymena mimbres.

    PubMed Central

    Zhang, X; Thompson, G A

    1997-01-01

    Sphingolipids are thought to stabilize glycosylphosphatidylinositol (GPI)-anchored protein-rich membrane domains of yeast and polarized higher animal cells during the processing and targeting of these proteins to the plasma membrane. A widely used criterion for identifying the stable sphingolipid- and GPI-anchored protein-enriched membrane domains is the resistance of these lipid-modified proteins to solubilization by the detergent Triton X-100 (TX-100) at low temperature. Surprisingly, there have been no reports of sphingolipid/GPI-anchored protein association in protozoans, despite the fact that these cells contain considerably higher levels of GPI-anchored proteins than does any other organism. We report here the presence in Tetrahymena mimbres of a significant pool of GPI-anchored proteins which resisted extraction by 1% TX-100 at 4 degrees C but not at 37 degrees C. Of the total cellular complement of GPI-anchored proteins, which together accounted for more than 2% of whole-cell protein and were especially enriched in surface membranes, 10% of the major 63kDa component (gpi63) and 23% of a somewhat less abundant component (gpi23) were insoluble in TX-100 at 4 degrees C. A substantial proportion of the cell's only abundant sphingolipid, ceramideaminoethylphosphonate (CAEP), was also insoluble in 1% TX-100 at 4 degrees C. Radiolabelling studies involving [3H]leucine incorporation into proteins and [3H]palmitic acid incorporation into lipids revealed that the TX-100-resistant gpi63, gpi23 and CAEP molecules were all metabolically distinct from their TX-100-soluble counterparts in other compartments of the cell. The presence of detergent-resistant sphingolipid/GPI-anchored protein domains in non-polarized ciliate and trypanosomatid cells was probably obscured in previous studies by the profusion of accompanying detergent-soluble molecules. PMID:9173882

  19. Serum sphingolipids: relationships to insulin sensitivity and changes with exercise in humans.

    PubMed

    Bergman, Bryan C; Brozinick, Joseph T; Strauss, Allison; Bacon, Samantha; Kerege, Anna; Bui, Hai Hoang; Sanders, Phil; Siddall, Parker; Kuo, Ming Shang; Perreault, Leigh

    2015-08-15

    Ceramides and sphingolipids are a family of lipid molecules that circulate in serum and accumulate in skeletal muscle, promoting insulin resistance. Plasma ceramide and dihydroceramide are related to insulin resistance, yet less is known regarding other ceramide and sphingolipid species. Despite its association with insulin sensitivity, chronic endurance exercise training does not change plasma ceramide and sphingolipid content, with little known regarding a single bout of exercise. We measured basal relationships and the effect of acute exercise (1.5 h at 50% V̇o2 max) and recovery on serum ceramide and sphingolipid content in sedentary obese individuals, endurance-trained athletes, and individuals with type 2 diabetes (T2D). Basal serum C18:0, C20:0, and C24:1 ceramide and C18:0 and total dihydroceramide were significantly higher in T2D and, along with C16:0 ceramide and C18:0 sphingomyelin, correlated positively with insulin resistance. Acute exercise significantly increased serum ceramide, glucosylceramide, and GM3 gangliosides, which largely decreased to basal values in recovery. Sphingosine 1-phosphate and sphingomyelin did not change during exercise but decreased below basal values in recovery. Serum C16:0 and C18:0 ceramide and C18:0 sphingomyelin, but not the total concentrations of either of them, were positively correlated with markers of muscle NF-κB activation, suggesting that specific species activate intracellular inflammation. Interestingly, a subset of sphingomyelin species, notably C14:0, C22:3, and C24:4 species, was positively associated with insulin secretion and glucose tolerance. Together, these data show that unique ceramide and sphingolipid species associate with either protective or deleterious features for diabetes and could provide novel therapeutic targets for the future.

  20. Lipopolysaccharide Disrupts Mitochondrial Physiology in Skeletal Muscle via Disparate Effects on Sphingolipid Metabolism.

    PubMed

    Hansen, Melissa E; Simmons, Kurtis J; Tippetts, Trevor S; Thatcher, Mikayla O; Saito, Rex R; Hubbard, Sheryl T; Trumbull, Annie M; Parker, Brian A; Taylor, Oliver J; Bikman, Benjamin T

    2015-12-01

    Lipopolysaccharides (LPS) are prevalent pathogenic molecules that are found within tissues and blood. Elevated circulating LPS is a feature of obesity and sepsis, both of which are associated with mitochondrial abnormalities that are key pathological features of LPS excess. However, the mechanism of LPS-induced mitochondrial alterations remains poorly understood. Herein we demonstrate the necessity of sphingolipid accrual in mediating altered mitochondrial physiology in skeletal muscle following LPS exposure. In particular, we found LPS elicited disparate effects on the sphingolipids dihydroceramides (DhCer) and ceramides (Cer) in both cultured myotubes and in muscle of LPS-injected mice. Although LPS-treated myotubes had reduced DhCer and increased Cer as well as increased mitochondrial respiration, muscle from LPS-injected mice manifested a reverse trend, namely elevated DhCer, but reduced Cer as well as reduced mitochondrial respiration. In addition, we found that LPS treatment caused mitochondrial fission, likely via dynamin-related protein 1, and increased oxidative stress. However, inhibition of de novo sphingolipid biosynthesis via myriocin protected normal mitochondrial function in spite of LPS, but inhibition of DhCer desaturase 1, which increases DhCer, but not Cer, exacerbated mitochondrial respiration with LPS. In an attempt to reconcile the incongruent effects of LPS in isolated muscle cells and whole muscle tissue, we incubated myotubes with conditioned medium from treated macrophages. In contrast to direct myotube LPS treatment, conditioned medium from LPS-treated macrophages reduced myotube respiration, but this was again mitigated with sphingolipid inhibition. Thus, macrophage sphingolipid production appears to be necessary for LPS-induced mitochondrial alterations in skeletal muscle tissue.

  1. Selective knockdown of ceramide synthases reveals complex interregulation of sphingolipid metabolism.

    PubMed

    Mullen, Thomas D; Spassieva, Stefka; Jenkins, Russell W; Kitatani, Kazuyuki; Bielawski, Jacek; Hannun, Yusuf A; Obeid, Lina M

    2011-01-01

    Mammalian ceramide synthases 1 to 6 (CerS1-6) generate Cer in an acyl-CoA-dependent manner, and expression of individual CerS has been shown to enhance the synthesis of ceramides with particular acyl chain lengths. However, the contribution of each CerS to steady-state levels of specific Cer species has not been evaluated. We investigated the knockdown of individual CerS in the MCF-7 human breast adenocarcinoma cell line by using small-interfering RNA (siRNA). We found that siRNA-induced downregulation of each CerS resulted in counter-regulation of nontargeted CerS. Additionally, each CerS knockdown produced unique effects on the levels of multiple sphingolipid species. For example, downregulation of CerS2 decreased very long-chain Cer but increased levels of CerS4, CerS5, and CerS6 expression and upregulated long-chain and medium-long-chain sphingolipids. Conversely, CerS6 knockdown decreased C16:0-Cer but increased CerS5 expression and caused non-C16:0 sphingolipids to be upregulated. Knockdown of individual CerS failed to decrease total sphingolipids or upregulate sphingoid bases. Treatment with siRNAs targeting combined CerS, CerS2, CerS5, and CerS6, did not change overall Cer or sphingomyelin mass but caused upregulation of dihydroceramide and hexosyl-ceramide and promoted endoplasmic reticulum stress. These data suggest that sphingolipid metabolism is robustly regulated by both redundancy in CerS-mediated Cer synthesis and counter-regulation of CerS expression.

  2. ARAP2 promotes GLUT1-mediated basal glucose uptake through regulation of sphingolipid metabolism.

    PubMed

    Chaudhari, Aditi; Håversen, Liliana; Mobini, Reza; Andersson, Linda; Ståhlman, Marcus; Lu, Emma; Rutberg, Mikael; Fogelstrand, Per; Ekroos, Kim; Mardinoglu, Adil; Levin, Malin; Perkins, Rosie; Borén, Jan

    2016-11-01

    Lipid droplet formation, which is driven by triglyceride synthesis, requires several droplet-associated proteins. We identified ARAP2 (an ADP-ribosylation factor 6 GTPase-activating protein) in the lipid droplet proteome of NIH-3T3 cells and showed that knockdown of ARAP2 resulted in decreased lipid droplet formation and triglyceride synthesis. We also showed that ARAP2 knockdown did not affect fatty acid uptake but reduced basal glucose uptake, total levels of the glucose transporter GLUT1, and GLUT1 levels in the plasma membrane and the lipid micro-domain fraction (a specialized plasma membrane domain enriched in sphingolipids). Microarray analysis showed that ARAP2 knockdown altered expression of genes involved in sphingolipid metabolism. Because sphingolipids are known to play a key role in cell signaling, we performed lipidomics to further investigate the relationship between ARAP2 and sphingolipids and potentially identify a link with glucose uptake. We found that ARAP2 knockdown increased glucosylceramide and lactosylceramide levels without affecting ceramide levels, and thus speculated that the rate-limiting enzyme in glycosphingolipid synthesis, namely glucosylceramide synthase (GCS), could be modified by ARAP2. In agreement with our hypothesis, we showed that the activity of GCS was increased by ARAP2 knockdown and reduced by ARAP2 overexpression. Furthermore, pharmacological inhibition of GCS resulted in increases in basal glucose uptake, total GLUT1 levels, triglyceride biosynthesis from glucose, and lipid droplet formation, indicating that the effects of GCS inhibition are the opposite to those resulting from ARAP2 knockdown. Taken together, our data suggest that ARAP2 promotes lipid droplet formation by modifying sphingolipid metabolism through GCS. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Alteration of complex sphingolipid composition and its physiological significance in yeast Saccharomyces cerevisiae lacking vacuolar ATPase.

    PubMed

    Tani, Motohiro; Toume, Moeko

    2015-12-01

    In the yeast Saccharomyces cerevisiae, complex sphingolipids have three types of polar head group and five types of ceramide; however, the physiological significance of the structural diversity is not fully understood. Here, we report that deletion of vacuolar H+-ATPase (V-ATPase) in yeast causes dramatic alteration of the complex sphingolipid composition, which includes decreases in hydroxylation at the C-4 position of long-chain bases and the C-2 position of fatty acids in the ceramide moiety, decreases in inositol phosphorylceramide (IPC) levels, and increases in mannosylinositol phosphorylceramide (MIPC) and mannosyldiinositol phosphorylceramide [M(IP)2C] levels. V-ATPase-deleted cells exhibited slow growth at pH 7.2, whereas the increase in MIPC levels was significantly enhanced when V-ATPase-deleted cells were incubated at pH 7.2. The protein expression levels of MIPC and M(IP)2C synthases were significantly increased in V-ATPase-deleted cells incubated at pH 7.2. Loss of MIPC synthesis or an increase in the hydroxylation level of the ceramide moiety of sphingolipids on overexpression of Scs7 and Sur2 sphingolipid hydroxylases enhanced the growth defect of V-ATPase-deleted cells at pH 7.2. On the contrary, the growth rate of V-ATPase-deleted cells was moderately increased on the deletion of SCS7 and SUR2. In addition, supersensitivities to Ca2+, Zn2+ and H2O2, which are typical phenotypes of V-ATPase-deleted cells, were enhanced by the loss of MIPC synthesis. These results indicate the possibility that alteration of the complex sphingolipid composition is an adaptation mechanism for a defect of V-ATPase.

  4. Yeast lacking the amphiphysin family protein Rvs167 is sensitive to disruptions in sphingolipid levels.

    PubMed

    Toume, Moeko; Tani, Motohiro

    2016-08-01

    Rvs167 and Rvs161 in Saccharomyces cerevisiae are amphiphysin family proteins, which are involved in several important cellular events, such as invagination and scission of endocytic vesicles, and actin cytoskeleton organization. It has been reported that cellular dysfunctions caused by deletion of RVS167 or RVS161 are rescued by deletion of specific nonessential sphingolipid-metabolizing enzyme genes. Here, we found that yeast cells lacking RVS167 or RVS161 exhibit a decrease in sphingolipid levels. In rvs167∆ cells, the expression level of Orm2, a negative regulator of serine palmitoyltransferase (SPT) catalyzing the initial step of sphingolipid biosynthesis, was increased in a calcineurin-dependent manner, and the decrease in sphingolipid levels in rvs167∆ cells was reversed on deletion of ORM2. Moreover, repression of both ORM1 and ORM2 expression or overexpression of SPT caused a strong growth defect of rvs167∆ cells, indicating that enhancement of de novo sphingolipid biosynthesis is detrimental to rvs167∆ cells. In contrast, partial repression of LCB1-encoding SPT suppressed abnormal phenotypes caused by the deletion of RVS167, including supersensitivity to high temperature and salt stress, and impairment of endocytosis and actin cytoskeleton organization. In addition, the partial repression of SPT activity suppressed the temperature supersensitivity and abnormal vacuolar morphology caused by deletion of VPS1 encoding a dynamin-like GTPase, which is required for vesicle scission and is functionally closely related to Rvs167/Rvs161, whereas repression of both ORM1 and ORM2 expression in vps1∆ cells caused a growth defect. Thus, it was suggested that proper regulation of SPT activity is indispensable for amphiphysin-deficient cells.

  5. Drug target identification in sphingolipid metabolism by computational systems biology tools: metabolic control analysis and metabolic pathway analysis.

    PubMed

    Ozbayraktar, F Betül Kavun; Ulgen, Kutlu O

    2010-08-01

    Sphingolipids regulate cellular processes that are critically important in cell's fate and function in cancer development and progression. This fact underlies the basics of the novel cancer therapy approach. The pharmacological manipulation of the sphingolipid metabolism in cancer therapeutics necessitates the detailed understanding of the pathway. Two computational systems biology tools are used to identify potential drug target enzymes among sphingolipid pathway that can be further utilized in drug design studies for cancer therapy. The enzymes in sphingolipid pathway were ranked according to their roles in controlling the metabolic network by metabolic control analysis. The physiologically connected reactions, i.e. biologically significant and functional modules of network, were identified by metabolic pathway analysis. The final set of candidate drug target enzymes are selected such that their manipulation leads to ceramide accumulation and long chain base phosphates depletion. The mathematical tools' efficiency for drug target identification performed in this study is validated by clinically available drugs. Copyright 2010 Elsevier Inc. All rights reserved.

  6. Genome-Wide Association Study Identifies Novel Loci Associated with Circulating Phospho- and Sphingolipid Concentrations

    PubMed Central

    Aulchenko, Yurii S.; Kirichenko, Anatoly V.; Janssens, A. Cecile J. W.; Jansen, Ritsert C.; Gnewuch, Carsten; Domingues, Francisco S.; Pattaro, Cristian; Wild, Sarah H.; Jonasson, Inger; Polasek, Ozren; Zorkoltseva, Irina V.; Hofman, Albert; Karssen, Lennart C.; Struchalin, Maksim; Floyd, James; Igl, Wilmar; Biloglav, Zrinka; Broer, Linda; Pfeufer, Arne; Pichler, Irene; Campbell, Susan; Zaboli, Ghazal; Kolcic, Ivana; Rivadeneira, Fernando; Huffman, Jennifer; Hastie, Nicholas D.; Uitterlinden, Andre; Franke, Lude; Franklin, Christopher S.; Vitart, Veronique; Nelson, Christopher P.; Preuss, Michael; Bis, Joshua C.; O'Donnell, Christopher J.; Franceschini, Nora; Witteman, Jacqueline C. M.; Axenovich, Tatiana; Oostra, Ben A.; Meitinger, Thomas; Hicks, Andrew A.; Hayward, Caroline; Wright, Alan F.; Gyllensten, Ulf; Campbell, Harry; Schmitz, Gerd

    2012-01-01

    Phospho- and sphingolipids are crucial cellular and intracellular compounds. These lipids are required for active transport, a number of enzymatic processes, membrane formation, and cell signalling. Disruption of their metabolism leads to several diseases, with diverse neurological, psychiatric, and metabolic consequences. A large number of phospholipid and sphingolipid species can be detected and measured in human plasma. We conducted a meta-analysis of five European family-based genome-wide association studies (N = 4034) on plasma levels of 24 sphingomyelins (SPM), 9 ceramides (CER), 57 phosphatidylcholines (PC), 20 lysophosphatidylcholines (LPC), 27 phosphatidylethanolamines (PE), and 16 PE-based plasmalogens (PLPE), as well as their proportions in each major class. This effort yielded 25 genome-wide significant loci for phospholipids (smallest P-value = 9.88×10−204) and 10 loci for sphingolipids (smallest P-value = 3.10×10−57). After a correction for multiple comparisons (P-value<2.2×10−9), we observed four novel loci significantly associated with phospholipids (PAQR9, AGPAT1, PKD2L1, PDXDC1) and two with sphingolipids (PLD2 and APOE) explaining up to 3.1% of the variance. Further analysis of the top findings with respect to within class molar proportions uncovered three additional loci for phospholipids (PNLIPRP2, PCDH20, and ABDH3) suggesting their involvement in either fatty acid elongation/saturation processes or fatty acid specific turnover mechanisms. Among those, 14 loci (KCNH7, AGPAT1, PNLIPRP2, SYT9, FADS1-2-3, DLG2, APOA1, ELOVL2, CDK17, LIPC, PDXDC1, PLD2, LASS4, and APOE) mapped into the glycerophospholipid and 12 loci (ILKAP, ITGA9, AGPAT1, FADS1-2-3, APOA1, PCDH20, LIPC, PDXDC1, SGPP1, APOE, LASS4, and PLD2) to the sphingolipid pathways. In large meta-analyses, associations between FADS1-2-3 and carotid intima media thickness, AGPAT1 and type 2 diabetes, and APOA1 and coronary artery disease were observed. In conclusion, our

  7. Genome-wide association study identifies novel loci associated with circulating phospho- and sphingolipid concentrations.

    PubMed

    Demirkan, Ayşe; van Duijn, Cornelia M; Ugocsai, Peter; Isaacs, Aaron; Pramstaller, Peter P; Liebisch, Gerhard; Wilson, James F; Johansson, Åsa; Rudan, Igor; Aulchenko, Yurii S; Kirichenko, Anatoly V; Janssens, A Cecile J W; Jansen, Ritsert C; Gnewuch, Carsten; Domingues, Francisco S; Pattaro, Cristian; Wild, Sarah H; Jonasson, Inger; Polasek, Ozren; Zorkoltseva, Irina V; Hofman, Albert; Karssen, Lennart C; Struchalin, Maksim; Floyd, James; Igl, Wilmar; Biloglav, Zrinka; Broer, Linda; Pfeufer, Arne; Pichler, Irene; Campbell, Susan; Zaboli, Ghazal; Kolcic, Ivana; Rivadeneira, Fernando; Huffman, Jennifer; Hastie, Nicholas D; Uitterlinden, Andre; Franke, Lude; Franklin, Christopher S; Vitart, Veronique; Nelson, Christopher P; Preuss, Michael; Bis, Joshua C; O'Donnell, Christopher J; Franceschini, Nora; Witteman, Jacqueline C M; Axenovich, Tatiana; Oostra, Ben A; Meitinger, Thomas; Hicks, Andrew A; Hayward, Caroline; Wright, Alan F; Gyllensten, Ulf; Campbell, Harry; Schmitz, Gerd

    2012-01-01

    Phospho- and sphingolipids are crucial cellular and intracellular compounds. These lipids are required for active transport, a number of enzymatic processes, membrane formation, and cell signalling. Disruption of their metabolism leads to several diseases, with diverse neurological, psychiatric, and metabolic consequences. A large number of phospholipid and sphingolipid species can be detected and measured in human plasma. We conducted a meta-analysis of five European family-based genome-wide association studies (N = 4034) on plasma levels of 24 sphingomyelins (SPM), 9 ceramides (CER), 57 phosphatidylcholines (PC), 20 lysophosphatidylcholines (LPC), 27 phosphatidylethanolamines (PE), and 16 PE-based plasmalogens (PLPE), as well as their proportions in each major class. This effort yielded 25 genome-wide significant loci for phospholipids (smallest P-value = 9.88×10(-204)) and 10 loci for sphingolipids (smallest P-value = 3.10×10(-57)). After a correction for multiple comparisons (P-value<2.2×10(-9)), we observed four novel loci significantly associated with phospholipids (PAQR9, AGPAT1, PKD2L1, PDXDC1) and two with sphingolipids (PLD2 and APOE) explaining up to 3.1% of the variance. Further analysis of the top findings with respect to within class molar proportions uncovered three additional loci for phospholipids (PNLIPRP2, PCDH20, and ABDH3) suggesting their involvement in either fatty acid elongation/saturation processes or fatty acid specific turnover mechanisms. Among those, 14 loci (KCNH7, AGPAT1, PNLIPRP2, SYT9, FADS1-2-3, DLG2, APOA1, ELOVL2, CDK17, LIPC, PDXDC1, PLD2, LASS4, and APOE) mapped into the glycerophospholipid and 12 loci (ILKAP, ITGA9, AGPAT1, FADS1-2-3, APOA1, PCDH20, LIPC, PDXDC1, SGPP1, APOE, LASS4, and PLD2) to the sphingolipid pathways. In large meta-analyses, associations between FADS1-2-3 and carotid intima media thickness, AGPAT1 and type 2 diabetes, and APOA1 and coronary artery disease were observed. In conclusion, our

  8. Plasma sphingolipids: potential biomarkers for severe hepatic fibrosis in chronic hepatitis C.

    PubMed

    Li, Jun-Feng; Qu, Feng; Zheng, Su-Jun; Ren, Feng; Wu, Hui-Li; Liu, Mei; Ren, Jin-Yu; Chen, Yu; Duan, Zhong-Ping; Zhang, Jin-Lan

    2015-07-01

    The plasma profile of sphingolipids in hepatic fibrosis patients with chronic hepatitis C (CHC) is rarely considered at present. The association between plasma sphingolipids and severe fibrosis in CHC remains an obscure area of research. The aim of the present study was to assess the plasma profile of sphingolipids and to examine the association between plasma sphingolipids and severe fibrosis in CHC, in order to identify potential novel markers of severe fibrosis in CHC. A cohort of 120 treatment-naïve patients with CHC were included in the present study. Liver biopsies were performed and routine serological indicators were measured. Plasma sphingolipids were detected using high performance liquid chromatography tandem mass spectrometry. A total of 44 plasma sphingolipids were detected. Plasma hexosylceramide (HexCer; d18:1/12:0), HexCer (d18:1/16:0) and HexCer (d18:1/22:0) were shown to be significantly different in patients with CHC between those with and without severe fibrosis (Metavir F ≥ 3; P < 0.05). HexCer (d18:1/12:0) was observed to be closely associated with severe fibrosis in CHC [odds ratio (OR)=1.03] following adjustment for confounding variables in a multivariate analysis. HexCer (d18:1/12:0) had diagnostic value for severe fibrosis in CHC [area under the curve (AUC)=0.69]. In patients with CHC who had developed significant fibrosis (Metavir F ≥ 2), HexCer (d18:1/12:0) remained closely associated with severe fibrosis (OR=1.08) in this subgroup. In addition, HexCer (d18:1/12:0) had sufficient diagnostic ability (AUC=0.73) to distinguish severe fibrosis in patients with CHC with significant fibrosis. In conclusion, the present study indicated that plasma HexCer (d18:1/12:0) exhibits a close correlation with severe hepatic fibrosis in CHC, particularly in patients who have significant fibrosis. Additionally, HexCer (d18:1/12:0) may be a potential marker of severe hepatic fibrosis in CHC.

  9. Segregation of sphingolipids and sterols during formation of secretory vesicles at the trans-Golgi network

    PubMed Central

    Klemm, Robin W.; Ejsing, Christer S.; Surma, Michal A.; Kaiser, Hermann-Josef; Gerl, Mathias J.; Sampaio, Julio L.; de Robillard, Quentin; Ferguson, Charles; Proszynski, Tomasz J.; Shevchenko, Andrej

    2009-01-01

    The trans-Golgi network (TGN) is the major sorting station in the secretory pathway of all eukaryotic cells. How the TGN sorts proteins and lipids to generate the enrichment of sphingolipids and sterols at the plasma membrane is poorly understood. To address this fundamental question in membrane trafficking, we devised an immunoisolation procedure for specific recovery of post-Golgi secretory vesicles transporting a transmembrane raft protein from the TGN to the cell surface in the yeast Saccharomyces cerevisiae. Using a novel quantitative shotgun lipidomics approach, we could demonstrate that TGN sorting selectively enriched ergosterol and sphingolipid species in the immunoisolated secretory vesicles. This finding, for the first time, indicates that the TGN exhibits the capacity to sort membrane lipids. Furthermore, the observation that the immunoisolated vesicles exhibited a higher membrane order than the late Golgi membrane, as measured by C-Laurdan spectrophotometry, strongly suggests that lipid rafts play a role in the TGN-sorting machinery. PMID:19433450

  10. Sphingolipids as Mediators in the Crosstalk between Microbiota and Intestinal Cells: Implications for Inflammatory Bowel Disease.

    PubMed

    Bryan, Phillips-Farfán; Karla, Carvajal; Edgar Alejandro, Medina-Torres; Sara Elva, Espinosa-Padilla; Gemma, Fabrias; Luz, Camacho

    Inflammatory bowel disease (IBD) describes different illnesses characterized by chronic inflammation of the gastrointestinal tract. Although the pathogenic mechanisms leading to IBD are poorly understood, immune system disturbances likely underlie its development. Sphingolipids (SLs) have been identified as important players and promising therapeutic targets to control inflammation in IBD. Interestingly, it seems that microorganisms of the normal gut microbiota and probiotics are involved in sphingolipid function. However, there is a great need to investigate the role of SLs as intermediates in the crosstalk between intestinal immunity and microorganisms. This review focuses on recent investigations that describe some mechanisms involved in the regulation of cytokine profiles by SLs. We also describe the importance of gut microbiota in providing signaling molecules that favor the communication between resident bacteria and intestinal cells. This, in turn, modulates the immune response in the bowel and likely in other peripheral organs. The potential of SLs and gut microbiota as targets or therapeutic agents for IBD is also discussed.

  11. Free-radical Destruction of Sphingolipids Resulting in 2-hexadecenal Formation

    PubMed Central

    Shadyro, Oleg; Lisovskaya, Alexandra; Semenkova, Galina; Edimecheva, Irina; Amaegberi, Nadezda

    2015-01-01

    The action of hypochlorous acid (HOCl) and γ-radiation on aqueous lysosphingolipid dispersions was found to produce 2-hexadecenal (Hex). This process includes the stages of formation of nitrogen-centered radicals from the starting molecules and the subsequent fragmentation of these radicals via the rupture of C–C and O–H bonds. These findings prove the existence of a nonenzymatic pathway of sphingolipid destruction leading to the formation of Hex, which possesses a wide spectrum of biological activity. Analysis of the effect of HOCl on transplantable rat glioma C6 cells and human embryonic kidney 293 cells points to the formation of Hex. This suggests that the described mechanism of free-radical destruction of sphingolipids may be replicated on cell culture under the stress of active chlorine forms. PMID:25861222

  12. Smart drugs for smarter stem cells: making SENSe (sphingolipid-enhanced neural stem cells) of ceramide.

    PubMed

    Bieberich, Erhard

    2008-01-01

    Ceramide and its derivative sphingosine-1-phosphate (S1P) are important signaling sphingolipids for neural stem cell apoptosis and differentiation. Most recently, our group has shown that novel ceramide analogs can be used to eliminate teratoma (stem cell tumor)-forming cells from a neural stem cell graft. In new studies, we found that S1P promotes survival of specific neural precursor cells that undergo differentiation to cells expressing oligodendroglial markers. Our studies suggest that a combination of novel ceramide and S1P analogs eliminates tumor-forming stem cells and at the same time, triggers oligodendroglial differentiation. This review discusses recent studies on the function of ceramide and S1P for the regulation of apoptosis, differentiation, and polarity in stem cells. We will also discuss results from ongoing studies in our laboratory on the use of sphingolipids in stem cell therapy. (c) 2008 S. Karger AG, Basel.

  13. Three-dimensional imaging of cholesterol and sphingolipids within a Madin-Darby canine kidney cell

    SciTech Connect

    Yeager, Ashley N.; Weber, Peter K.; Kraft, Mary L.

    2016-01-08

    Metabolic stable isotope incorporation and secondary ion mass spectrometry(SIMS) depth profiling performed on a Cameca NanoSIMS 50 were used to image the 18O-cholesterol and 15N-sphingolipid distributions within a portion of a Madin-Darby canine kidney (MDCK) cell. Three-dimensional representations of the component-specific isotope distributions show clearly defined regions of 18O-cholesterol and 15N-sphingolipid enrichment that seem to be separate subcellular compartments. Furthermore, the low levels of nitrogen-containing secondary ions detected at the 18O-enriched regions suggest that these 18O-cholesterol-rich structures may be lipiddroplets, which have a core consisting of cholesterol esters and triacylglycerides.

  14. Three-dimensional imaging of cholesterol and sphingolipids within a Madin-Darby canine kidney cell

    DOE PAGES

    Yeager, Ashley N.; Weber, Peter K.; Kraft, Mary L.

    2016-01-08

    Metabolic stable isotope incorporation and secondary ion mass spectrometry(SIMS) depth profiling performed on a Cameca NanoSIMS 50 were used to image the 18O-cholesterol and 15N-sphingolipid distributions within a portion of a Madin-Darby canine kidney (MDCK) cell. Three-dimensional representations of the component-specific isotope distributions show clearly defined regions of 18O-cholesterol and 15N-sphingolipid enrichment that seem to be separate subcellular compartments. Furthermore, the low levels of nitrogen-containing secondary ions detected at the 18O-enriched regions suggest that these 18O-cholesterol-rich structures may be lipiddroplets, which have a core consisting of cholesterol esters and triacylglycerides.

  15. Plasma Phospholipid and Sphingolipid Alterations in Presenilin1 Mutation Carriers: A Pilot Study.

    PubMed

    Chatterjee, Pratishtha; Lim, Wei L F; Shui, Guanghou; Gupta, Veer B; James, Ian; Fagan, Anne M; Xiong, Chengjie; Sohrabi, Hamid R; Taddei, Kevin; Brown, Belinda M; Benzinger, Tammie; Masters, Colin; Snowden, Stuart G; Wenk, Marcus R; Bateman, Randall J; Morris, John C; Martins, Ralph N

    2016-01-01

    Aberrant lipid metabolism has been implicated in sporadic Alzheimer's disease (AD). The current study investigated plasma phospholipid and sphingolipid profiles in individuals carrying PSEN1 mutations responsible for autosomal dominant AD (ADAD). Study participants evaluated were from the Perth and Melbourne sites of the Dominantly Inherited Alzheimer Network (DIAN) study. Plasma phospholipid and sphingolipid profiles were measured using liquid chromatography coupled with mass spectrometry in 20 PSEN1 mutation carriers (MC; eight of whom were symptomatic and twelve asymptomatic, based on Clinical Dementia Rating scores) and compared with six non carriers (NC) using linear mixed models. Further, AD gold standard biomarker data obtained from the DIAN database were correlated with lipid species significantly altered between MC and NC, using Spearman's correlation coefficient. One-hundred and thirty-nine plasma phospholipid and sphingolipid species were measured. Significantly altered species in MC compared to NC primarily belonged to choline and ethanolamine containing phospholipid classes and ceramides. Further phosphatidylcholine species (34:6, 36:5, 40:6) correlated with cerebrospinal fluid tau (p <  0.05), and plasmalogen ethanolamine species (34:2, 36:,4) correlated with both cerebrospinal fluid tau and brain amyloid load within the MC group (p <  0.05). These findings indicate altered phospholipid and sphingolipid metabolism in ADAD and provide insight into the pathomolecular changes occurring with ADAD pathogenesis. Further, findings reported in this study allow comparison of lipid alterations in ADAD with those reported previously in sporadic AD. The findings observed in the current pilot study warrant validation in the larger DIAN cohort.

  16. Alterations of calcium homoeostasis in cultured rat astrocytes evoked by bioactive sphingolipids.

    PubMed

    Stenovec, M; Trkov, S; Kreft, M; Zorec, R

    2014-09-01

    In the brain, alterations in sphingolipid metabolism contribute to several neurological disorders; however, their effect on astrocytes is largely unknown. Here, we identified bioactive sphingolipids that affect intracellular free calcium concentration ([Ca(2+)]i), mobility of peptidergic secretory vesicles, signalling pathways involved in alterations of calcium homoeostasis and explored the relationship between the stimulus-evoked increase in [Ca(2+)]i and attenuation of vesicle mobility. Confocal time-lapse images were acquired to explore [Ca(2+)]i signals, the mobility of fluorescently tagged peptidergic vesicles and the structural integrity of the microtubules and actin filaments before and after the addition of exogenous sphingolipids to astrocytes. Fingolimod (FTY720), a recently introduced therapeutic for multiple sclerosis, and sphingosine, a releasable constituent of membrane sphingolipids, evoked long-lasting increases in [Ca(2+)]i in the presence and absence of extracellular Ca(2+); the evoked responses were diminished in the absence of extracellular Ca(2+). Activation of phospholipase C and inositol-1,4,5-triphosphate receptors was necessary and sufficient to evoke increases in [Ca(2+)]i as revealed by the pharmacologic inhibitors; Ca(2+) flux from the extracellular space intensified these responses several fold. The lipid-evoked increases in [Ca(2+)]i coincided with the attenuated vesicle mobility. High and positive correlation between increase in [Ca(2+)]i and decrease in peptidergic vesicle mobility was confirmed independently in astrocytes exposed to evoked, transient Ca(2+) signalling triggered by purinergic and glutamatergic stimulation. Exogenously added cell-permeable sphingosine-like lipids exert complex, Ca(2+)-dependent effects on astrocytes and likely alter their homeostatic function in vivo. © 2014 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.

  17. Sphingolipid Metabolism Correlates with Cerebrospinal Fluid Beta Amyloid Levels in Alzheimer’s Disease

    PubMed Central

    Fonteh, Alfred N.; Ormseth, Cora; Chiang, Jiarong; Cipolla, Matthew; Arakaki, Xianghong; Harrington, Michael G.

    2015-01-01

    Sphingolipids are important in many brain functions but their role in Alzheimer’s disease (AD) is not completely defined. A major limit is availability of fresh brain tissue with defined AD pathology. The discovery that cerebrospinal fluid (CSF) contains abundant nanoparticles that include synaptic vesicles and large dense core vesicles offer an accessible sample to study these organelles, while the supernatant fluid allows study of brain interstitial metabolism. Our objective was to characterize sphingolipids in nanoparticles representative of membrane vesicle metabolism, and in supernatant fluid representative of interstitial metabolism from study participants with varying levels of cognitive dysfunction. We recently described the recruitment, diagnosis, and CSF collection from cognitively normal or impaired study participants. Using liquid chromatography tandem mass spectrometry, we report that cognitively normal participants had measureable levels of sphingomyelin, ceramide, and dihydroceramide species, but that their distribution differed between nanoparticles and supernatant fluid, and further differed in those with cognitive impairment. In CSF from AD compared with cognitively normal participants: a) total sphingomyelin levels were lower in nanoparticles and supernatant fluid; b) levels of ceramide species were lower in nanoparticles and higher in supernatant fluid; c) three sphingomyelin species were reduced in the nanoparticle fraction. Moreover, three sphingomyelin species in the nanoparticle fraction were lower in mild cognitive impairment compared with cognitively normal participants. The activity of acid, but not neutral sphingomyelinase was significantly reduced in the CSF from AD participants. The reduction in acid sphingomylinase in CSF from AD participants was independent of depression and psychotropic medications. Acid sphingomyelinase activity positively correlated with amyloid β42 concentration in CSF from cognitively normal but not impaired

  18. Sphingolipids metabolism in the salivary glands of rats with obesity and streptozotocin induced diabetes

    PubMed Central

    Garbowska, Marta; Mikłosz, Agnieszka; Wróblewski, Igor; Kurek, Krzysztof; Ostrowska, Lucyna; Chabowski, Adrian; Żendzian‐Piotrowska, Małgorzata; Zalewska, Anna

    2017-01-01

    Diabetes is considered a major public health problem affecting millions of individuals worldwide. Remarkably, scientific reports regarding salivary glands sphingolipid metabolism in diabetes are virtually non‐existent. This is odd given the well‐established link between the both in other tissues (e.g., skeletal muscles, liver) and the key role of these glands in oral health preservation. The aim of this paper is to examine sphingolipids metabolism in the salivary glands in (pre)diabetes (evoked by high fat diet feeding or streptozotocin). Wistar rats were allocated into three groups: control, HFD‐, or STZ‐diabetes. The content of major sphingolipid classes in the parotid (PSG) and submandibular (SMSG) glands was assessed via chromatography. Additionally, Western blot analyses were employed for the evaluation of key sphingolipid signaling pathway enzyme levels. No changes in ceramide content in the PSG were found, whereas an increase in ceramide concentration for SMSG of the STZ group was observed. This was accompanied by an elevation in SPT1 level. Probably also sphingomyelin hydrolysis was increased in the SMSG of the STZ‐diabetic rats, since we observed a significant drop in the amount of SM. PSG and SMSG respond differently to (pre)diabetes, with clearer pattern presented by the later gland. An activation of sphingomyelin signaling pathway was observed in the course of STZ‐diabetes, that is, metabolic condition with rapid onset/progression. Whereas, chronic HFD lead to an inhibition of sphingomyelin signaling pathway in the salivary glands (manifested in an inhibition of ceramide de novo synthesis and accumulation of S1P). PMID:28369933

  19. Sphingolipids metabolism in the salivary glands of rats with obesity and streptozotocin induced diabetes.

    PubMed

    Garbowska, Marta; Łukaszuk, Bartłomiej; Mikłosz, Agnieszka; Wróblewski, Igor; Kurek, Krzysztof; Ostrowska, Lucyna; Chabowski, Adrian; Żendzian-Piotrowska, Małgorzata; Zalewska, Anna

    2017-10-01

    Diabetes is considered a major public health problem affecting millions of individuals worldwide. Remarkably, scientific reports regarding salivary glands sphingolipid metabolism in diabetes are virtually non-existent. This is odd given the well-established link between the both in other tissues (e.g., skeletal muscles, liver) and the key role of these glands in oral health preservation. The aim of this paper is to examine sphingolipids metabolism in the salivary glands in (pre)diabetes (evoked by high fat diet feeding or streptozotocin). Wistar rats were allocated into three groups: control, HFD-, or STZ-diabetes. The content of major sphingolipid classes in the parotid (PSG) and submandibular (SMSG) glands was assessed via chromatography. Additionally, Western blot analyses were employed for the evaluation of key sphingolipid signaling pathway enzyme levels. No changes in ceramide content in the PSG were found, whereas an increase in ceramide concentration for SMSG of the STZ group was observed. This was accompanied by an elevation in SPT1 level. Probably also sphingomyelin hydrolysis was increased in the SMSG of the STZ-diabetic rats, since we observed a significant drop in the amount of SM. PSG and SMSG respond differently to (pre)diabetes, with clearer pattern presented by the later gland. An activation of sphingomyelin signaling pathway was observed in the course of STZ-diabetes, that is, metabolic condition with rapid onset/progression. Whereas, chronic HFD lead to an inhibition of sphingomyelin signaling pathway in the salivary glands (manifested in an inhibition of ceramide de novo synthesis and accumulation of S1P). © 2017 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.

  20. Fenofibrate lowers atypical sphingolipids in plasma of dyslipidemic patients: A novel approach for treating diabetic neuropathy?

    PubMed

    Othman, Alaa; Benghozi, Renee; Alecu, Irina; Wei, Yu; Niesor, Eric; von Eckardstein, Arnold; Hornemann, Thorsten

    2015-01-01

    The condensation of palmitoyl-CoA and L-Serine is the first step in the de novo formation of sphingolipids and catalyzed by the serine-palmitoyltransferase (SPT). Besides other acyl-CoAs the SPT can also metabolize L-alanine and glycine, which forms an atypical category of neurotoxic 1-deoxy-sphingolipids (1-deoxySL). Several mutations in SPT are associated with pathologically increased 1-deoxySL levels, which cause the inherited sensory neuropathy HSAN1. 1-DeoxySL levels are also elevated in individuals with the metabolic syndrome and diabetes mellitus type II and seem to be involved in the pathology of the diabetic neuropathy. In previous studies, we observed a strong correlation between plasma 1-deoxySLs and triglycerides (TGs). We were therefore interested whether lowering plasma TG levels also affects plasma sphingolipid and in particular, 1-deoxySL levels. Sixty-six patients with dyslipidemia were treated for 6 wk with the TG-lowering drug fenofibrate (160 mg/d) or extended-release niacin (0.5 g/d for 3 wk, then 1 g/d) with 4 wk of washout between treatments. The sphingoid base profile was analyzed by liquid chromatography-mass spectrometry (LC-MS) before and after each treatment block. Fenofibrate significantly lowered 1-deoxySLs and other atypical sphingoid bases (P < .001) but had no effect on the typical sphingolipids. In contrast, extended-release niacin had no effect on 1-deoxySL levels although both treatments lowered plasma TG levels. The lowering of plasma 1-deoxySL levels by fenofibrate in dyslipidemic patients might be a novel therapeutic approach in the prevention and treatment of diabetic neuropathy. Copyright © 2015 National Lipid Association. Published by Elsevier Inc. All rights reserved.

  1. Iron, glucose and intrinsic factors alter sphingolipid composition as yeast cells enter stationary phase.

    PubMed

    Lester, Robert L; Withers, Bradley R; Schultz, Megan A; Dickson, Robert C

    2013-04-01

    Survival of Saccharomyces cerevisiae cells, like most microorganisms, requires switching from a rapidly dividing to a non-dividing or stationary state. To further understand how cells navigate this switch, we examined sphingolipids since they are key structural elements of membranes and also regulate signaling pathways vital for survival. During and after the switch to a non-dividing state there is a large increase in total free and sphingolipid-bound long chain-bases and an even larger increase in free and bound C20-long-chain bases, which are nearly undetectable in dividing cells. These changes are due to intrinsic factors including Orm1 and Orm2, ceramide synthase, Lcb4 kinase and the Tsc3 subunit of serine palmitoyltransferase as well as extrinsic factors including glucose and iron. Lowering the concentration of glucose, a form of calorie restriction, decreases the level of LCBs, which is consistent with the idea that reducing the level of some sphingolipids enhances lifespan. In contrast, iron deprivation increases LCB levels and decreases long term survival; however, these phenomena may not be related because iron deprivation disrupts many metabolic pathways. The correlation between increased LCBs and shorter lifespan is unsupported at this time. The physiological rise in LCBs that we observe may serve to modulate nutrient transporters and possibly other membrane phenomena that contribute to enhanced stress resistance and survival in stationary phase. Copyright © 2013 Elsevier B.V. All rights reserved.

  2. Glycosylation of inositol phosphorylceramide sphingolipids is required for normal growth and reproduction in Arabidopsis

    SciTech Connect

    Tartaglio, Virginia; Rennie, Emilie A.; Cahoon, Rebecca; Wang, George; Baidoo, Edward; Mortimer, Jennifer C.; Cahoon, Edgar B.; Scheller, Henrik V.

    2016-09-19

    Sphingolipids are a major component of plant plasma membranes and endomembranes, and mediate a diverse range of biological processes. Study of the highly glycosylated glycosyl inositol phosphorylceramide (GIPC) sphingolipids has been slow as a result of challenges associated with the extractability of GIPCs, and their functions in the plant remain poorly characterized. We recently discovered an Arabidopsis GIPC glucuronosyltransferase, INOSITOL PHOSPHORYLCERAMIDE GLUCURONOSYLTRANSFERASE 1 (IPUT1), which is the first enzyme in the GIPC glycosylation pathway. Plants homozygous for the iput1 loss-of-function mutation were unobtainable, and so the developmental effects of reduced GIPC glucuronosylation could not be analyzed in planta. Using a pollen-specific rescue construct, we have here isolated homozygous iput1 mutants. The iput1 mutants show severe dwarfism, compromised pollen tube guidance, and constitutive activation of salicyclic acid-mediated defense pathways. The mutants also possess reduced GIPCs, increased ceramides, and an increased incorporation of short-chain fatty acids and dihydroxylated bases into inositol phosphorylceramides and GIPCs. The assignment of a direct role for GIPC glycan head groups in the impaired processes in iput1 mutants is complicated by the vast compensatory changes in the sphingolipidome; however, our results reveal that the glycosylation steps of GIPC biosynthesis are important regulated components of sphingolipid metabolism. In conclusion, this study corroborates previously suggested roles for GIPC glycans in plant growth and defense, suggests important role s for them in reproduction and demonstrates that the entire sphingolipidome is sensitive to their status.

  3. PLP-dependent enzymes as entry and exit gates of sphingolipid metabolism

    PubMed Central

    Bourquin, Florence; Capitani, Guido; Grütter, Markus Gerhard

    2011-01-01

    Sphingolipids are membrane constituents as well as signaling molecules involved in many essential cellular processes. Serine palmitoyltransferase (SPT) and sphingosine-1-phosphate lyase (SPL), both PLP (pyridoxal 5′-phosphate)-dependent enzymes, function as entry and exit gates of the sphingolipid metabolism. SPT catalyzes the condensation of serine and a fatty acid into 3-keto-dihydrosphingosine, whereas SPL degrades sphingosine-1-phosphate (S1P) into phosphoethanolamine and a long-chain aldehyde. The recently solved X-ray structures of prokaryotic homologs of SPT and SPL combined with functional studies provide insight into the structure–function relationship of the two enzymes. Despite carrying out different reactions, the two enzymes reveal striking similarities in the overall fold, topology, and residues crucial for activity. Unlike their eukaryotic counterparts, bacterial SPT and SPL lack a transmembrane helix, making them targets of choice for biochemical characterization because the use of detergents can be avoided. Both human enzymes are linked to severe diseases or disorders and might therefore serve as targets for the development of therapeutics aiming at the modulation of their activity. This review gives an overview of the sphingolipid metabolism and of the available biochemical studies of prokaryotic SPT and SPL, and discusses the major similarities and differences to the corresponding eukaryotic enzymes. PMID:21710479

  4. Iron, Glucose and Intrinsic Factors Alter Sphingolipid Composition as Yeast Cells Enter Stationary Phase

    PubMed Central

    Lester, Robert L.; Withers, Bradley R.; Schultz, Megan A.; Dickson, Robert C.

    2013-01-01

    Survival of Saccharomyces cerevisiae cells, like most microorganisms, requires switching from a rapidly dividing to a non-dividing or stationary state. To further understand how cells navigate this switch, we examined sphingolipids since they are key structural elements of membranes and also regulate signaling pathways vital for survival. During and after the switch to a non-dividing state there is a large increase in total free and sphingolipid-bound long chain-bases and an even larger increase in free and bound C20-long-chain bases, which are nearly undetectable in dividing cells. These changes are due to intrinsic factors including Orm1 and Orm2, ceramide synthase, Lcb4 kinase and the Tsc3 subunit of serine palmitoyltransferase as well as extrinsic factors including glucose and iron. Lowering the concentration of glucose, a form of calorie restriction, decreases the level of LCBs, which is consistent with the idea that reducing the level of some sphingolipids enhances lifespan. In contrast, iron deprivation increases LCB levels and decreases long term survival; however, these phenomena may not be related because iron deprivation disrupts many metabolic pathways. The correlation between increased LCBs and shorter lifespan is unsupported at this time. The physiological rise in LCBs that we observe may serve to modulate nutrient transporters and possibly other membrane phenomena that contribute to enhanced stress resistance and survival in stationary phase. PMID:23286903

  5. Multiplex analysis of sphingolipids using amine-reactive tags (iTRAQ).

    PubMed

    Nabetani, Takuji; Makino, Asami; Hullin-Matsuda, Françoise; Hirakawa, Taka-Aki; Takeoka, Shinji; Okino, Nozomu; Ito, Makoto; Kobayashi, Toshihide; Hirabayashi, Yoshio

    2011-06-01

    Ceramides play a crucial role in divergent signaling events, including differentiation, senescence, proliferation, and apoptosis. Ceramides are a minor lipid component in terms of content; thus, highly sensitive detection is required for accurate quantification. The recently developed isobaric tags for relative and absolute quantitation (iTRAQ) method enables a precise comparison of both protein and aminophospholipids. However, iTRAQ tagging had not been applied to the determination of sphingolipids. Here we report a method for the simultaneous measurement of multiple ceramide and monohexosylceramide samples using iTRAQ tags. Samples were hydrolyzed with sphingolipid ceramide N-deacylase (SCDase) to expose the free amino group of the sphingolipids, to which the N-hydroxysuccinimide group of iTRAQ reagent was conjugated. The reaction was performed in the presence of a cleavable detergent, 3-[3-(1,1-bisalkyloxyethyl)pyridine-1-yl]propane-1-sulfonate (PPS) to both improve the hydrolysis and ensure the accuracy of the mass spectrometry analysis performed after iTRAQ labeling. This method was successfully applied to the profiling of ceramides and monohexosylceramides in sphingomyelinase-treated Madin Darby canine kidney (MDCK) cells and apoptotic Jurkat cells.

  6. Multiplex analysis of sphingolipids using amine-reactive tags (iTRAQ)

    PubMed Central

    Nabetani, Takuji; Makino, Asami; Hullin-Matsuda, Françoise; Hirakawa, Taka-aki; Takeoka, Shinji; Okino, Nozomu; Ito, Makoto; Kobayashi, Toshihide; Hirabayashi, Yoshio

    2011-01-01

    Ceramides play a crucial role in divergent signaling events, including differentiation, senescence, proliferation, and apoptosis. Ceramides are a minor lipid component in terms of content; thus, highly sensitive detection is required for accurate quantification. The recently developed isobaric tags for relative and absolute quantitation (iTRAQ) method enables a precise comparison of both protein and aminophospholipids. However, iTRAQ tagging had not been applied to the determination of sphingolipids. Here we report a method for the simultaneous measurement of multiple ceramide and monohexosylceramide samples using iTRAQ tags. Samples were hydrolyzed with sphingolipid ceramide N-deacylase (SCDase) to expose the free amino group of the sphingolipids, to which the N-hydroxysuccinimide group of iTRAQ reagent was conjugated. The reaction was performed in the presence of a cleavable detergent, 3-[3-(1,1-bisalkyloxyethyl)pyridine-1-yl]propane-1-sulfonate (PPS) to both improve the hydrolysis and ensure the accuracy of the mass spectrometry analysis performed after iTRAQ labeling. This method was successfully applied to the profiling of ceramides and monohexosylceramides in sphingomyelinase-treated Madin Darby canine kidney (MDCK) cells and apoptotic Jurkat cells. PMID:21487068

  7. CRS-MIS in Candida glabrata: sphingolipids modulate echinocandin-Fks interaction.

    PubMed

    Healey, Kelley R; Katiyar, Santosh K; Raj, Shriya; Edlind, Thomas D

    2012-10-01

    Infections with the azole-refractory yeast Candida glabrata are now commonly treated with the echinocandins caspofungin (CSF) or micafungin (MCF). True resistance (>  32-fold decreased susceptibility) to these lipopeptide inhibitors of cell wall synthesis is rare and strictly associated with mutations in integral membrane proteins Fks1 or Fks2. In contrast, mutants exhibiting 4- to 32-fold CSF reduced susceptibility (CRS) were readily selected in vitro, and surprisingly demonstrated 4- to 32-fold MCF increased susceptibility (MIS). Sequencing and gene deletion demonstrated that CRS-MIS is Fks-independent. To explore alternative mechanisms, we initially employed Saccharomyces cerevisiae, and observed that CRS was conferred by multiple mutations (fen1Δ, sur4Δ, cka2Δ and tsc10-ts) disrupting sphingolipid biosynthesis. Following this lead, C. glabrata fen1Δ and cka2Δ deletants were constructed, and shown to exhibit CRS-MIS. Sphingolipid analysis of CRS-MIS laboratory mutants and clinical isolates demonstrated elevated dihydrosphingosine (DHS) and phytosphingosine (PHS) levels, and consistent with this sequencing revealed fen1, sur4, ifa38 and sur2 mutations. Moreover, exogenous DHS or PHS conferred a CRS-MIS phenotype on wild-type C. glabrata. Exogenous PHS failed, however, to suppress CRS-MIS in a sur2 mutant blocked in conversion of DHS to PHS, implying that accumulation of these intermediates confers CRS-MIS. We conclude that membrane sphingolipids modulate echinocandin-Fks interaction. © 2012 Blackwell Publishing Ltd.

  8. Plasma Membrane Proteolipid 3 Protein Modulates Amphotericin B Resistance through Sphingolipid Biosynthetic Pathway

    PubMed Central

    Bari, Vinay K.; Sharma, Sushma; Alfatah, Md.; Mondal, Alok K.; Ganesan, K.

    2015-01-01

    Invasive opportunistic fungal infections of humans are common among those suffering from impaired immunity, and are difficult to treat resulting in high mortality. Amphotericin B (AmB) is one of the few antifungals available to treat such infections. The AmB resistance mechanisms reported so far mainly involve decrease in ergosterol content or alterations in cell wall. In contrast, depletion of sphingolipids sensitizes cells to AmB. Recently, overexpression of PMP3 gene, encoding plasma membrane proteolipid 3 protein, was shown to increase and its deletion to decrease, AmB resistance. Here we have explored the mechanistic basis of PMP3 effect on AmB resistance. It was found that ergosterol content and cell wall integrity are not related to modulation of AmB resistance by PMP3. A few prominent phenotypes of PMP3 delete strain, namely, defective actin polarity, impaired salt tolerance, and reduced rate of endocytosis are also not related to its AmB-sensitivity. However, PMP3 overexpression mediated increase in AmB resistance requires a functional sphingolipid pathway. Moreover, AmB sensitivity of strains deleted in PMP3 can be suppressed by the addition of phytosphingosine, a sphingolipid pathway intermediate, confirming the importance of this pathway in modulation of AmB resistance by PMP3. PMID:25965669

  9. Rapid nanoscale quantitative analysis of plant sphingolipid long-chain bases by GC-MS.

    PubMed

    Cacas, Jean-Luc; Melser, Su; Domergue, Frédéric; Joubès, Jérôme; Bourdenx, Brice; Schmitter, Jean-Marie; Mongrand, Sébastien

    2012-07-01

    In eukaryotic organisms, sphingolipids are major structural lipids of biological membranes and perform additional essential functions as signalling molecules. While long-chain bases (LCB), the common precursor to all sphingolipid classes, is represented by only one major molecular species in animals and fungi, up to nine LCB have been found in plants. In the absence of genuine plant sphingolipid references required for proper quantification, we have reinvestigated and optimized a protocol destined to the quantification of total plant LCB that relies on the use of gas chromatography-mass spectrometry (GC-MS). This rapid three-step protocol sequentially involves (1) the release of LCB from biological samples using barium hydroxide solution, (2) their oxidation into aldehydes by metaperiodate, and (3) the subsequent identification/quantification of these aldehydes by GC-MS. It is simple and reliable and enables separation of aldehydes upon their stero-specificity. It further enables the quantification of total LCB from a wide variety of samples including yeast and animal cell cultures.

  10. Endothelial Nogo-B regulates sphingolipid biosynthesis to promote pathological cardiac hypertrophy during chronic pressure overload

    PubMed Central

    Huang, Yan; Azevedo, Paula S.; Siragusa, Mauro; Bielawski, Jacek; Giordano, Frank J.

    2016-01-01

    We recently discovered that endothelial Nogo-B, a membrane protein of the ER, regulates vascular function by inhibiting the rate-limiting enzyme, serine palmitoyltransferase (SPT), in de novo sphingolipid biosynthesis. Here, we show that endothelium-derived sphingolipids, particularly sphingosine-1-phosphate (S1P), protect the heart from inflammation, fibrosis, and dysfunction following pressure overload and that Nogo-B regulates this paracrine process. SPT activity is upregulated in banded hearts in vivo as well as in TNF-α–activated endothelium in vitro, and loss of Nogo removes the brake on SPT, increasing local S1P production. Hence, mice lacking Nogo-B, systemically or specifically in the endothelium, are resistant to the onset of pathological cardiac hypertrophy. Furthermore, pharmacological inhibition of SPT with myriocin restores permeability, inflammation, and heart dysfunction in Nogo-A/B–deficient mice to WT levels, whereas SEW2871, an S1P1 receptor agonist, prevents myocardial permeability, inflammation, and dysfunction in WT banded mice. Our study identifies a critical role of endothelial sphingolipid biosynthesis and its regulation by Nogo-B in the development of pathological cardiac hypertrophy and proposes a potential therapeutic target for the attenuation or reversal of this clinical condition. PMID:27158676

  11. Loss of Frataxin activates the iron/sphingolipid/PDK1/Mef2 pathway in mammals

    PubMed Central

    Chen, Kuchuan; Ho, Tammy Szu-Yu; Lin, Guang; Tan, Kai Li; Rasband, Matthew N; Bellen, Hugo J

    2016-01-01

    Friedreich’s ataxia (FRDA) is an autosomal recessive neurodegenerative disease caused by mutations in Frataxin (FXN). Loss of FXN causes impaired mitochondrial function and iron homeostasis. An elevated production of reactive oxygen species (ROS) was previously proposed to contribute to the pathogenesis of FRDA. We recently showed that loss of frataxin homolog (fh), a Drosophila homolog of FXN, causes a ROS independent neurodegeneration in flies (Chen et al., 2016). In fh mutants, iron accumulation in the nervous system enhances the synthesis of sphingolipids, which in turn activates 3-phosphoinositide dependent protein kinase-1 (Pdk1) and myocyte enhancer factor-2 (Mef2) to trigger neurodegeneration of adult photoreceptors. Here, we show that loss of Fxn in the nervous system in mice also activates an iron/sphingolipid/PDK1/Mef2 pathway, indicating that the mechanism is evolutionarily conserved. Furthermore, sphingolipid levels and PDK1 activity are also increased in hearts of FRDA patients, suggesting that a similar pathway is affected in FRDA. DOI: http://dx.doi.org/10.7554/eLife.20732.001 PMID:27901468

  12. Sphingolipid metabolism is strikingly different between pollen and leaf in Arabidopsis as revealed by compositional and gene expression profiling.

    PubMed

    Luttgeharm, Kyle D; Kimberlin, Athen N; Cahoon, Rebecca E; Cerny, Ronald L; Napier, Johnathan A; Markham, Jonathan E; Cahoon, Edgar B

    2015-07-01

    Although sphingolipids are essential for male gametophytic development in Arabidopsis thaliana, sphingolipid composition and biosynthetic gene expression have not been previously examined in pollen. In this report, electrospray ionization (ESI)-MS/MS was applied to characterization of sphingolipid compositional profiles in pollen isolated from wild type Arabidopsis Col-0 and a long-chain base (LCB) Δ4 desaturase mutant. Pollen fractions were highly enriched in glucosylceramides (GlcCer) relative to levels previously reported in leaves. Accompanying the loss of the Δ4 unsaturated LCB sphingadiene (d18:2) in the Δ4 desaturase mutant was a 50% reduction in GlcCer concentrations. In addition, pollen glycosylinositolphosphoceramides (GIPCs) were found to have a complex array of N-acetyl-glycosylated GIPCs, including species with up to three pentose units that were absent from leaf GIPCs. Underlying the distinct sphingolipid composition of pollen, genes for key biosynthetic enzymes for GlcCer and d18:2 synthesis and metabolism were more highly expressed in pollen than in leaves or seedlings, including genes for GlcCer synthase (GCS), sphingoid base C-4 hydroxylase 2 (SBH2), LCB Δ8 desaturases (SLD1 and SLD2), and LOH2 ceramide synthase (LOH2). Overall, these findings indicate strikingly divergent sphingolipid metabolism between pollen and leaves in Arabidopsis, the significance of which remains to be determined.

  13. Glycosylation of inositol phosphorylceramide sphingolipids is required for normal growth and reproduction in Arabidopsis.

    PubMed

    Tartaglio, Virginia; Rennie, Emilie A; Cahoon, Rebecca; Wang, George; Baidoo, Edward; Mortimer, Jennifer C; Cahoon, Edgar B; Scheller, Henrik V

    2017-01-01

    Sphingolipids are a major component of plant plasma membranes and endomembranes, and mediate a diverse range of biological processes. Study of the highly glycosylated glycosyl inositol phosphorylceramide (GIPC) sphingolipids has been slow as a result of challenges associated with the extractability of GIPCs, and their functions in the plant remain poorly characterized. We recently discovered an Arabidopsis GIPC glucuronosyltransferase, INOSITOL PHOSPHORYLCERAMIDE GLUCURONOSYLTRANSFERASE 1 (IPUT1), which is the first enzyme in the GIPC glycosylation pathway. Plants homozygous for the iput1 loss-of-function mutation were unobtainable, and so the developmental effects of reduced GIPC glucuronosylation could not be analyzed in planta. Using a pollen-specific rescue construct, we have here isolated homozygous iput1 mutants. The iput1 mutants show severe dwarfism, compromised pollen tube guidance, and constitutive activation of salicyclic acid-mediated defense pathways. The mutants also possess reduced GIPCs, increased ceramides, and an increased incorporation of short-chain fatty acids and dihydroxylated bases into inositol phosphorylceramides and GIPCs. The assignment of a direct role for GIPC glycan head groups in the impaired processes in iput1 mutants is complicated by the vast compensatory changes in the sphingolipidome; however, our results reveal that the glycosylation steps of GIPC biosynthesis are important regulated components of sphingolipid metabolism. This study corroborates previously suggested roles for GIPC glycans in plant growth and defense, suggests important roles for them in reproduction and demonstrates that the entire sphingolipidome is sensitive to their status. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

  14. Disruption of sphingolipid biosynthesis in hepatocyte nodules: selective proliferative stimulus induced by fumonisin B1.

    PubMed

    van der Westhuizen, Liana; Gelderblom, Wentzel C A; Shephard, Gordon S; Swanevelder, Sonja

    2004-07-15

    In order to investigate the role of sphingolipid disruption in the cancer promoting potential of fumonisin B(1) (FB(1)) in the development of hepatocyte nodules, male Fischer 344 rats were subjected to cancer initiation (FB(1) containing diet or diethylnitrosamine (DEN) by i.p. injection) and promotion (2-acetylaminofluorene with partial hepatectomy, 2-AAF/PH) treatments followed by a secondary FB(1) dietary regimen. Sphinganine (Sa) and sphingosine (So) levels were measured by high performance liquid chromatography in control, surrounding and nodular liver tissues of the rats. The disruption of sphingolipid biosynthesis by the secondary FB(1) treatment in the control rats was significantly (P < 0.05) enhanced by the 2-AAF/PH cancer promotion treatment. The nodular and surrounding Sa levels returned to baseline following FB(1) initiation and 2-AAF/PH promotion. When comparing the groups subjected to the secondary FB(1) treatment, the initiation effected by FB(1) was less (P < 0.01) sensitive to the accumulation of Sa in the nodular and surrounding tissues than DEN initiation and the 2-AAF/PH control treatment. In contrast, the So level of FB(1) initiation was marginally increased in the nodules compared to the surrounding liver after 2-AAF/PH promotion and significantly (P < 0.05) higher with the secondary FB(1) treatment. Although, the FB(1)-induced hepatocyte nodules were not resistant to the disruption of sphingolipid biosynthesis, the nodular So levels were increased and might provide a selective growth stimulus possibly induced by bio-active sphingoid intermediates such as sphingosine 1-phosphate (S1P).

  15. Characterization of Drosophila Saposin-related mutants as a model for lysosomal sphingolipid storage diseases

    PubMed Central

    Schulze, Heike; Paradis, Marie; Gosejacob, Dominic; Papan, Cyrus; Shevchenko, Andrej; Psathaki, Olympia Ekaterina; Thielisch, Melanie; Sandhoff, Konrad

    2017-01-01

    ABSTRACT Sphingolipidoses are inherited diseases belonging to the class of lysosomal storage diseases (LSDs), which are characterized by the accumulation of indigestible material in the lysosome caused by specific defects in the lysosomal degradation machinery. While some LSDs can be efficiently treated by enzyme replacement therapy (ERT), this is not possible if the nervous system is affected due to the presence of the blood-brain barrier. Sphingolipidoses in particular often present as severe, untreatable forms of LSDs with massive sphingolipid and membrane accumulation in lysosomes, neurodegeneration and very short life expectancy. The digestion of intralumenal membranes within lysosomes is facilitated by lysosomal sphingolipid activator proteins (saposins), which are cleaved from a prosaposin precursor. Prosaposin mutations cause some of the severest forms of sphingolipidoses, and are associated with perinatal lethality in mice, hampering studies on disease progression. We identify the Drosophila prosaposin orthologue Saposin-related (Sap-r) as a key regulator of lysosomal lipid homeostasis in the fly. Its mutation leads to a typical spingolipidosis phenotype with an enlarged endolysosomal compartment and sphingolipid accumulation as shown by mass spectrometry and thin layer chromatography. Sap-r mutants show reduced viability with ∼50% survival to adulthood, allowing us to study progressive neurodegeneration and analyze their lipid profile in young and aged flies. Additionally, we observe a defect in sterol homeostasis with local sterol depletion at the plasma membrane. Furthermore, we find that autophagy is increased, resulting in the accumulation of mitochondria in lysosomes, concomitant with increased oxidative stress. Together, we establish Drosophila Sap-r mutants as a lysosomal storage disease model suitable for studying the age-dependent progression of lysosomal dysfunction associated with lipid accumulation and the resulting pathological signaling

  16. Sphingolipids: Key Regulators of Apoptosis and Pivotal Players in Cancer Drug Resistance

    PubMed Central

    Giussani, Paola; Tringali, Cristina; Riboni, Laura; Viani, Paola; Venerando, Bruno

    2014-01-01

    Drug resistance elicited by cancer cells still constitutes a huge problem that frequently impairs the efficacy of both conventional and novel molecular therapies. Chemotherapy usually acts to induce apoptosis in cancer cells; therefore, the investigation of apoptosis control and of the mechanisms used by cancer cells to evade apoptosis could be translated in an improvement of therapies. Among many tools acquired by cancer cells to this end, the de-regulated synthesis and metabolism of sphingolipids have been well documented. Sphingolipids are known to play many structural and signalling roles in cells, as they are involved in the control of growth, survival, adhesion, and motility. In particular, in order to increase survival, cancer cells: (a) counteract the accumulation of ceramide that is endowed with pro-apoptotic potential and is induced by many drugs; (b) increase the synthesis of sphingosine-1-phosphate and glucosylceramide that are pro-survivals signals; (c) modify the synthesis and the metabolism of complex glycosphingolipids, particularly increasing the levels of modified species of gangliosides such as 9-O acetylated GD3 (αNeu5Ac(2-8)αNeu5Ac(2-3)βGal(1-4)βGlc(1-1)Cer) or N-glycolyl GM3 (αNeu5Ac (2-3)βGal(1-4)βGlc(1-1)Cer) and de-N-acetyl GM3 (NeuNH(2)βGal(1-4)βGlc(1-1)Cer) endowed with anti-apoptotic roles and of globoside Gb3 related to a higher expression of the multidrug resistance gene MDR1. In light of this evidence, the employment of chemical or genetic approaches specifically targeting sphingolipid dysregulations appears a promising tool for the improvement of current chemotherapy efficacy. PMID:24625663

  17. Evaluation of Sphingolipids in Wistar Rats Treated to Prolonged and Single Oral Doses of Fumonisin B1

    PubMed Central

    Direito, Glória M.; Almeida, Adriana P.; Aquino, Simone; dos Reis, Tatiana Alves; Pozzi, Claudia Rodrigues; Corrêa, Benedito

    2009-01-01

    The objective of the present study was to evaluate sphingolipid levels (sphingosine-So and sphinganine-Sa) and to compare the Sa/So ratio in liver, serum and urine of Wistar rats after prolonged administration (21 days) of fumonisin B1 (FB1). In parallel, the kinetics of sphingolipid elimination in urine was studied in animals receiving a single dose of FB1. Prolonged exposure to FB1 caused an increase in Sa levels in urine, serum and liver. The most marked effect on sphingolipid biosynthesis was observed in animals treated with the highest dose of FB1. Animals receiving a single dose of FB1 presented variations in Sa and So levels and in the Sa/So ratio. PMID:19333435

  18. Analysis of Mammalian Sphingolipids by Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) and Tissue Imaging Mass Spectrometry (TIMS)

    PubMed Central

    Sullards, M. Cameron; Liu, Ying; Chen, Yanfeng; Merrill, Alfred H.

    2011-01-01

    Sphingolipids are a highly diverse category of molecules that serve not only as components of biological structures but also as regulators of numerous cell functions. Because so many of the structural features of sphingolipids give rise to their biological activity, there is a need for comprehensive or “sphingolipidomic” methods for identification and quantitation of as many individual subspecies as possible. This review defines sphingolipids as a class, briefly discusses classical methods for their analysis, and focuses primarily on liquid chromatography tandem mass spectrometry (LC-MS/MS) and tissue imaging mass spectrometry (TIMS). Recently, a set of evolving and expanding methods have been developed and rigorously validated for the extraction, identification, separation, and quantitation of sphingolipids by LC-MS/MS. Quantitation of these biomolecules is made possible via the use of an internal standard cocktail. The compounds that can be readily analyzed are free long-chain (sphingoid) bases, sphingoid base 1-phosphates, and more complex species such as ceramides, ceramide 1-phosphates, sphingomyelins, mono- and di-hexosylceramides sulfatides, and novel compounds such as the 1-deoxy- and 1-(deoxymethyl)-sphingoid bases and their N-acyl-derivatives. These methods can be altered slightly to separate and quantitate isomeric species such as glucosyl/galactosylceramide. Because these techniques require the extraction of sphingolipids from their native environment, any information regarding their localization in histological slices is lost. Therefore, this review also describes methods for TIMS. This technique has been shown to be a powerful tool to determine the localization of individual molecular species of sphingolipids directly from tissue slices. PMID:21749933

  19. Localization of Sphingolipid Enriched Plasma Membrane Regions and Long-Chain Base Composition during Mature-Fruit Abscission in Olive

    PubMed Central

    Parra-Lobato, Maria C.; Paredes, Miguel A.; Labrador, Juana; Saucedo-García, Mariana; Gavilanes-Ruiz, Marina; Gomez-Jimenez, Maria C.

    2017-01-01

    Sphingolipids, found in membranes of eukaryotic cells, have been demonstrated to carry out functions in various processes in plant cells. However, the roles of these lipids in fruit abscission remain to be determined in plants. Biochemical and fluorescence microscopy imaging approach has been adopted to investigate the accumulation and distribution of sphingolipids during mature-fruit abscission in olive (Olea europaea L. cv. Picual). Here, a lipid-content analysis in live protoplasts of the olive abscission zone (AZ) was made with fluorescent dyes and lipid analogs, particularly plasma membrane sphingolipid-enriched domains, and their dynamics were investigated in relation to the timing of mature-fruit abscission. In olive AZ cells, the measured proportion of both polar lipids and sphingolipids increased as well as endocytosis was stimulated during mature-fruit abscission. Likewise, mature-fruit abscission resulted in quantitative and qualitative changes in sphingolipid long-chain bases (LCBs) in the olive AZ. The total LCB increase was due essentially to the increase of t18:1(8E) LCBs, suggesting that C-4 hydroxylation and Δ8 desaturation with a preference for (E)-isomer formation were quantitatively the most important sphingolipids in olive AZ during abscission. However, our results also showed a specific association between the dihydroxylated LCB sphinganine (d18:0) and the mature-fruit abscission. These results indicate a clear correlation between the sphingolipid composition and mature-fruit abscission. Moreover, measurements of endogenous sterol levels in the olive AZ revealed that it accumulated sitosterol and campesterol with a concomitant decrease in cycloartenol during abscission. In addition, underlying the distinct sterol composition of AZ during abscission, genes for key biosynthetic enzymes for sterol synthesis, for obtusifoliol 14α-demethylase (CYP51) and C-24 sterol methyltransferase2 (SMT2), were up-regulated during mature-fruit abscission, in

  20. Localization of Sphingolipid Enriched Plasma Membrane Regions and Long-Chain Base Composition during Mature-Fruit Abscission in Olive.

    PubMed

    Parra-Lobato, Maria C; Paredes, Miguel A; Labrador, Juana; Saucedo-García, Mariana; Gavilanes-Ruiz, Marina; Gomez-Jimenez, Maria C

    2017-01-01

    Sphingolipids, found in membranes of eukaryotic cells, have been demonstrated to carry out functions in various processes in plant cells. However, the roles of these lipids in fruit abscission remain to be determined in plants. Biochemical and fluorescence microscopy imaging approach has been adopted to investigate the accumulation and distribution of sphingolipids during mature-fruit abscission in olive (Olea europaea L. cv. Picual). Here, a lipid-content analysis in live protoplasts of the olive abscission zone (AZ) was made with fluorescent dyes and lipid analogs, particularly plasma membrane sphingolipid-enriched domains, and their dynamics were investigated in relation to the timing of mature-fruit abscission. In olive AZ cells, the measured proportion of both polar lipids and sphingolipids increased as well as endocytosis was stimulated during mature-fruit abscission. Likewise, mature-fruit abscission resulted in quantitative and qualitative changes in sphingolipid long-chain bases (LCBs) in the olive AZ. The total LCB increase was due essentially to the increase of t18:1(8E) LCBs, suggesting that C-4 hydroxylation and Δ8 desaturation with a preference for (E)-isomer formation were quantitatively the most important sphingolipids in olive AZ during abscission. However, our results also showed a specific association between the dihydroxylated LCB sphinganine (d18:0) and the mature-fruit abscission. These results indicate a clear correlation between the sphingolipid composition and mature-fruit abscission. Moreover, measurements of endogenous sterol levels in the olive AZ revealed that it accumulated sitosterol and campesterol with a concomitant decrease in cycloartenol during abscission. In addition, underlying the distinct sterol composition of AZ during abscission, genes for key biosynthetic enzymes for sterol synthesis, for obtusifoliol 14α-demethylase (CYP51) and C-24 sterol methyltransferase2 (SMT2), were up-regulated during mature-fruit abscission, in

  1. Involvement of complex sphingolipids and phosphatidylserine in endosomal trafficking in yeast Saccharomyces cerevisiae.

    PubMed

    Tani, Motohiro; Kuge, Osamu

    2012-12-01

    Sphingolipids play critical roles in many physiologically important events in the yeast Saccharomyces cerevisiae. In this study, we found that csg2Δ mutant cells defective in the synthesis of mannosylinositol phosphorylceramide exhibited abnormal intracellular accumulation of an exocytic v-SNARE, Snc1, under phosphatidylserine synthase gene (PSS1)-repressive conditions, although in wild-type cells, Snc1 was known to cycle between plasma membranes and the late Golgi via post-Golgi endosomes. The mislocalized Snc1 was co-localized with an endocytic marker dye, FM4-64, upon labelling for a short time. The abnormal distribution of Snc1 was suppressed by deletion of GYP2 encoding a GTPase-activating protein that negatively regulates endosomal vesicular trafficking, or expression of GTP-restricted form of Ypt32 GTPase. Furthermore, an endocytosis-deficient mutant of Snc1 was localized to plasma membranes in PSS1-repressed csg2Δ mutant cells as well as wild-type cells. Thus, the PSS1-repressed csg2Δ mutant cells were indicated to be defective in the trafficking of Snc1 from post-Golgi endosomes to the late Golgi. In contrast, the vesicular trafficking pathways via pre-vacuolar endosomes in the PSS1-repressed csg2Δ mutant cells seemed to be normal. These results suggested that specific complex sphingolipids and phosphatidylserine are co-ordinately involved in specific vesicular trafficking pathway. © 2012 Blackwell Publishing Ltd.

  2. Identification of a sphingolipid α-glucuronosyltransferase that is essential for pollen function in Arabidopsis.

    PubMed

    Rennie, Emilie A; Ebert, Berit; Miles, Godfrey P; Cahoon, Rebecca E; Christiansen, Katy M; Stonebloom, Solomon; Khatab, Hoda; Twell, David; Petzold, Christopher J; Adams, Paul D; Dupree, Paul; Heazlewood, Joshua L; Cahoon, Edgar B; Scheller, Henrik Vibe

    2014-08-01

    Glycosyl inositol phosphorylceramide (GIPC) sphingolipids are a major class of lipids in fungi, protozoans, and plants. GIPCs are abundant in the plasma membrane in plants, comprising around a quarter of the total lipids in these membranes. Plant GIPCs contain unique glycan decorations that include a conserved glucuronic acid (GlcA) residue and various additional sugars; however, no proteins responsible for glycosylating GIPCs have been identified to date. Here, we show that the Arabidopsis thaliana protein INOSITOL PHOSPHORYLCERAMIDE GLUCURONOSYLTRANSFERASE1 (IPUT1) transfers GlcA from UDP-GlcA to GIPCs. To demonstrate IPUT1 activity, we introduced the IPUT1 gene together with genes for a UDP-glucose dehydrogenase from Arabidopsis and a human UDP-GlcA transporter into a yeast mutant deficient in the endogenous inositol phosphorylceramide (IPC) mannosyltransferase. In this engineered yeast strain, IPUT1 transferred GlcA to IPC. Overexpression or silencing of IPUT1 in Nicotiana benthamiana resulted in an increase or a decrease, respectively, in IPC glucuronosyltransferase activity in vitro. Plants in which IPUT1 was silenced accumulated IPC, the immediate precursor, as well as ceramides and glucosylceramides. Plants overexpressing IPUT1 showed an increased content of GIPCs. Mutations in IPUT1 are not transmitted through pollen, indicating that these sphingolipids are essential in plants. © 2014 American Society of Plant Biologists. All rights reserved.

  3. Loss of Inositol Phosphorylceramide Sphingolipid Mannosylation Induces Plant Immune Responses and Reduces Cellulose Content in Arabidopsis

    SciTech Connect

    Fang, Lin; Ishikawa, Toshiki; Rennie, Emilie A.; Murawska, Gosia M.; Lao, Jeemeng; Yan, Jingwei; Tsai, Alex Yi-Lin; Baidoo, Edward E. K.; Xu, Jun; Keasling, Jay D.; Demura, Taku; Kawai-Yamada, Maki; Scheller, Henrik V.; Mortimer, Jenny C.

    2016-11-28

    Glycosylinositol phosphorylceramides (GIPCs) are a class of glycosylated sphingolipids found in plants, fungi, and protozoa. These lipids are abundant in the plant plasma membrane, forming ~25% of total plasma membrane lipids. Little is known about the function of the glycosylated headgroup, but two recent studies have indicated that they play a key role in plant signaling and defense. Here, we show that a member of glycosyltransferase family 64, previously named ECTOPICALLY PARTING CELLS1, is likely a Golgi-localized GIPC-specific mannosyl-transferase, which we renamed GIPC MANNOSYL-TRANSFERASE1 (GMT1). Sphingolipid analysis revealed that the Arabidopsis thaliana gmt1 mutant almost completely lacks mannose-carrying GIPCs. Heterologous expression of GMT1 in Saccharomyces cerevisiae and tobacco (Nicotiana tabacum) cv Bright Yellow 2 resulted in the production of non-native mannosylated GIPCs. gmt1 displays a severe dwarfed phenotype and a constitutive hypersensitive response characterized by elevated salicylic acid and hydrogen peroxide levels, similar to that we previously reported for the Golgi-localized, GIPC-specific, GDP-Man transporter GONST1 (Mortimer et al., 2013). Unexpectedly, we show that gmt1 cell walls have a reduction in cellulose content, although other matrix polysaccharides are unchanged.

  4. Loss of Inositol Phosphorylceramide Sphingolipid Mannosylation Induces Plant Immune Responses and Reduces Cellulose Content in Arabidopsis.

    PubMed

    Fang, Lin; Ishikawa, Toshiki; Rennie, Emilie A; Murawska, Gosia M; Lao, Jeemeng; Yan, Jingwei; Tsai, Alex Yi-Lin; Baidoo, Edward E K; Xu, Jun; Keasling, Jay D; Demura, Taku; Kawai-Yamada, Maki; Scheller, Henrik V; Mortimer, Jenny C

    2016-12-01

    Glycosylinositol phosphorylceramides (GIPCs) are a class of glycosylated sphingolipids found in plants, fungi, and protozoa. These lipids are abundant in the plant plasma membrane, forming ∼25% of total plasma membrane lipids. Little is known about the function of the glycosylated headgroup, but two recent studies have indicated that they play a key role in plant signaling and defense. Here, we show that a member of glycosyltransferase family 64, previously named ECTOPICALLY PARTING CELLS1, is likely a Golgi-localized GIPC-specific mannosyl-transferase, which we renamed GIPC MANNOSYL-TRANSFERASE1 (GMT1). Sphingolipid analysis revealed that the Arabidopsis thaliana gmt1 mutant almost completely lacks mannose-carrying GIPCs. Heterologous expression of GMT1 in Saccharomyces cerevisiae and tobacco (Nicotiana tabacum) cv Bright Yellow 2 resulted in the production of non-native mannosylated GIPCs. gmt1 displays a severe dwarfed phenotype and a constitutive hypersensitive response characterized by elevated salicylic acid and hydrogen peroxide levels, similar to that we previously reported for the Golgi-localized, GIPC-specific, GDP-Man transporter GONST1 (Mortimer et al., 2013). Unexpectedly, we show that gmt1 cell walls have a reduction in cellulose content, although other matrix polysaccharides are unchanged. © 2016 American Society of Plant Biologists. All rights reserved.

  5. Loss of Inositol Phosphorylceramide Sphingolipid Mannosylation Induces Plant Immune Responses and Reduces Cellulose Content in Arabidopsis

    DOE PAGES

    Fang, Lin; Ishikawa, Toshiki; Rennie, Emilie A.; ...

    2016-11-28

    Glycosylinositol phosphorylceramides (GIPCs) are a class of glycosylated sphingolipids found in plants, fungi, and protozoa. These lipids are abundant in the plant plasma membrane, forming ~25% of total plasma membrane lipids. Little is known about the function of the glycosylated headgroup, but two recent studies have indicated that they play a key role in plant signaling and defense. Here, we show that a member of glycosyltransferase family 64, previously named ECTOPICALLY PARTING CELLS1, is likely a Golgi-localized GIPC-specific mannosyl-transferase, which we renamed GIPC MANNOSYL-TRANSFERASE1 (GMT1). Sphingolipid analysis revealed that the Arabidopsis thaliana gmt1 mutant almost completely lacks mannose-carrying GIPCs. Heterologousmore » expression of GMT1 in Saccharomyces cerevisiae and tobacco (Nicotiana tabacum) cv Bright Yellow 2 resulted in the production of non-native mannosylated GIPCs. gmt1 displays a severe dwarfed phenotype and a constitutive hypersensitive response characterized by elevated salicylic acid and hydrogen peroxide levels, similar to that we previously reported for the Golgi-localized, GIPC-specific, GDP-Man transporter GONST1 (Mortimer et al., 2013). Unexpectedly, we show that gmt1 cell walls have a reduction in cellulose content, although other matrix polysaccharides are unchanged.« less

  6. A procedure for fractionation of sphingolipid classes by solid-phase extraction on aminopropyl cartridges.

    PubMed

    Bodennec, J; Koul, O; Aguado, I; Brichon, G; Zwingelstein, G; Portoukalian, J

    2000-09-01

    Solid-phase extraction (SPE) methods are easy, rapid, and reliable. Their growing popularity is in part due to their operational simplicity and cost reduction in solvents, and partly because they are easier to automate. Sphingolipids are implicated in various cellular events such as growth, differentiation, and apoptosis. However, their separation by small SPE cartridges has attracted limited attention. Here we describe an SPE procedure on aminopropyl cartridges that by sequential elution allows the separation of a lipid mixture into free ceramides, neutral glycosphingolipids, neutral phospholipids (sphingomyelin), and a fraction containing the acidic phospholipids and phosphorylated sphingoid bases, phosphoceramides and sulfatides. Individual components are obtained in high yield and purity. We applied the procedure to obtain data on separation of [(3)H]myristic acid-labeled sphingolipids from fish gills, and from human melanoma tumor tissue. Individual lipids in the SPE fractions were identified by chromatography on several high-performance thin-layer chromatography (HPTLC) systems. The chromatographic behavior of free sphingoid bases is also reported.

  7. Legionella pneumophila S1P-lyase targets host sphingolipid metabolism and restrains autophagy.

    PubMed

    Rolando, Monica; Escoll, Pedro; Nora, Tamara; Botti, Joëlle; Boitez, Valérie; Bedia, Carmen; Daniels, Craig; Abraham, Gilu; Stogios, Peter J; Skarina, Tatiana; Christophe, Charlotte; Dervins-Ravault, Delphine; Cazalet, Christel; Hilbi, Hubert; Rupasinghe, Thusitha W T; Tull, Dedreia; McConville, Malcolm J; Ong, Sze Ying; Hartland, Elizabeth L; Codogno, Patrice; Levade, Thierry; Naderer, Thomas; Savchenko, Alexei; Buchrieser, Carmen

    2016-02-16

    Autophagy is an essential component of innate immunity, enabling the detection and elimination of intracellular pathogens. Legionella pneumophila, an intracellular pathogen that can cause a severe pneumonia in humans, is able to modulate autophagy through the action of effector proteins that are translocated into the host cell by the pathogen's Dot/Icm type IV secretion system. Many of these effectors share structural and sequence similarity with eukaryotic proteins. Indeed, phylogenetic analyses have indicated their acquisition by horizontal gene transfer from a eukaryotic host. Here we report that L. pneumophila translocates the effector protein sphingosine-1 phosphate lyase (LpSpl) to target the host sphingosine biosynthesis and to curtail autophagy. Our structural characterization of LpSpl and its comparison with human SPL reveals high structural conservation, thus supporting prior phylogenetic analysis. We show that LpSpl possesses S1P lyase activity that was abrogated by mutation of the catalytic site residues. L. pneumophila triggers the reduction of several sphingolipids critical for macrophage function in an LpSpl-dependent and -independent manner. LpSpl activity alone was sufficient to prevent an increase in sphingosine levels in infected host cells and to inhibit autophagy during macrophage infection. LpSpl was required for efficient infection of A/J mice, highlighting an important virulence role for this effector. Thus, we have uncovered a previously unidentified mechanism used by intracellular pathogens to inhibit autophagy, namely the disruption of host sphingolipid biosynthesis.

  8. Sphingolipids from a symbiotic microbe regulate homeostasis of host intestinal natural killer T cells

    PubMed Central

    An, Dingding; Oh, Sungwhan F.; Olszak, Torsten; Neves, Joana F.; Avci, Fikri; Erturk-Hasdemir, Deniz; Lu, Xi; Zeissig, Sebastian; Blumberg, Richard S.; Kasper, Dennis L.

    2014-01-01

    Summary Co-evolution of beneficial microorganisms with the mammalian intestine fundamentally shapes mammalian physiology. Herein we report that the intestinal microbe Bacteroides fragilis modifies the homeostasis of host invariant natural killer T (iNKT) cells by supplementing the host’s endogenous lipid antigen milieu with unique inhibitory sphingolipids. The process occurs early in life and effectively impedes iNKT cell proliferation during neonatal development. Consequently, total colonic iNKT cell numbers are restricted into adulthood and hosts are protected against experimental iNKT cell–mediated, oxazolone-induced colitis. In studies with neonatal mice lacking access to bacterial sphingolipids, we found that treatment with B. fragilis glycosphingolipids—exemplified by an isolated peak (M.W.=717.6) called GSL-Bf717—reduces colonic iNKT cell numbers and confers protection against oxazolone-induced colitis in adulthood. Our results suggest that the distinctive inhibitory capacity of GSL-Bf717 and similar molecules may prove useful in the treatment of autoimmune and allergic disorders in which iNKT cell activation is destructive. PMID:24439373

  9. Functional identification of a delta8-sphingolipid desaturase from Borago officinalis.

    PubMed

    Sperling, P; Libisch, B; Zähringer, U; Napier, J A; Heinz, E

    2001-04-15

    The similarities between delta12- and delta5-fatty acyl desaturase sequences were used to construct degenerate primers for PCR experiments with cDNA transcribed from mRNA of developing borage seeds. Screening of a borage seed cDNA library with an amplified DNA fragment resulted in the isolation of a full-length cDNA corresponding to a deduced open-reading frame of 446 amino acids. The protein showed high similarity to plant delta8-sphingolipid desaturases as well as to the delta6-fatty acyl desaturase from Borago officinalis. The sequence is characterized by the presence of a N-terminal cytochrome b5 domain. Expression of this open-reading frame in Saccharomyces cerevisiae resulted in the formation of delta8-trans/cis-phytosphingenines not present in wild-type cells, as shown by HPLC analysis of sphingoid bases as their dinitrophenyl derivatives. GLC-MS analysis of the methylated di-O-trimethylsilyl ether derivatives confirmed the presence of delta8-stereoisomers of C18- and C20-phytosphingenine. Furthermore, Northern blotting showed that the gene encoding a stereo-unselective delta8-sphingolipid desaturase is primarily expressed in young borage leaves.

  10. Sphingolipids as Mediators in the Crosstalk between Microbiota and Intestinal Cells: Implications for Inflammatory Bowel Disease

    PubMed Central

    Bryan, Phillips-Farfán; Edgar Alejandro, Medina-Torres; Sara Elva, Espinosa-Padilla; Gemma, Fabrias

    2016-01-01

    Inflammatory bowel disease (IBD) describes different illnesses characterized by chronic inflammation of the gastrointestinal tract. Although the pathogenic mechanisms leading to IBD are poorly understood, immune system disturbances likely underlie its development. Sphingolipids (SLs) have been identified as important players and promising therapeutic targets to control inflammation in IBD. Interestingly, it seems that microorganisms of the normal gut microbiota and probiotics are involved in sphingolipid function. However, there is a great need to investigate the role of SLs as intermediates in the crosstalk between intestinal immunity and microorganisms. This review focuses on recent investigations that describe some mechanisms involved in the regulation of cytokine profiles by SLs. We also describe the importance of gut microbiota in providing signaling molecules that favor the communication between resident bacteria and intestinal cells. This, in turn, modulates the immune response in the bowel and likely in other peripheral organs. The potential of SLs and gut microbiota as targets or therapeutic agents for IBD is also discussed. PMID:27656050

  11. Legionella pneumophila S1P-lyase targets host sphingolipid metabolism and restrains autophagy

    PubMed Central

    Rolando, Monica; Escoll, Pedro; Nora, Tamara; Botti, Joëlle; Boitez, Valérie; Daniels, Craig; Abraham, Gilu; Stogios, Peter J.; Skarina, Tatiana; Christophe, Charlotte; Dervins-Ravault, Delphine; Cazalet, Christel; Hilbi, Hubert; Rupasinghe, Thusitha W. T.; Tull, Dedreia; McConville, Malcolm J.; Ong, Sze Ying; Hartland, Elizabeth L.; Codogno, Patrice; Levade, Thierry; Naderer, Thomas; Savchenko, Alexei; Buchrieser, Carmen

    2016-01-01

    Autophagy is an essential component of innate immunity, enabling the detection and elimination of intracellular pathogens. Legionella pneumophila, an intracellular pathogen that can cause a severe pneumonia in humans, is able to modulate autophagy through the action of effector proteins that are translocated into the host cell by the pathogen’s Dot/Icm type IV secretion system. Many of these effectors share structural and sequence similarity with eukaryotic proteins. Indeed, phylogenetic analyses have indicated their acquisition by horizontal gene transfer from a eukaryotic host. Here we report that L. pneumophila translocates the effector protein sphingosine-1 phosphate lyase (LpSpl) to target the host sphingosine biosynthesis and to curtail autophagy. Our structural characterization of LpSpl and its comparison with human SPL reveals high structural conservation, thus supporting prior phylogenetic analysis. We show that LpSpl possesses S1P lyase activity that was abrogated by mutation of the catalytic site residues. L. pneumophila triggers the reduction of several sphingolipids critical for macrophage function in an LpSpl-dependent and -independent manner. LpSpl activity alone was sufficient to prevent an increase in sphingosine levels in infected host cells and to inhibit autophagy during macrophage infection. LpSpl was required for efficient infection of A/J mice, highlighting an important virulence role for this effector. Thus, we have uncovered a previously unidentified mechanism used by intracellular pathogens to inhibit autophagy, namely the disruption of host sphingolipid biosynthesis. PMID:26831115

  12. A sphingolipid-dependent diffusion barrier confines ER stress to the yeast mother cell

    PubMed Central

    Clay, Lori; Caudron, Fabrice; Denoth-Lippuner, Annina; Boettcher, Barbara; Buvelot Frei, Stéphanie; Snapp, Erik Lee; Barral, Yves

    2014-01-01

    In many cell types, lateral diffusion barriers compartmentalize the plasma membrane and, at least in budding yeast, the endoplasmic reticulum (ER). However, the molecular nature of these barriers, their mode of action and their cellular functions are unclear. Here, we show that misfolded proteins of the ER remain confined into the mother compartment of budding yeast cells. Confinement required the formation of a lateral diffusion barrier in the form of a distinct domain of the ER-membrane at the bud neck, in a septin-, Bud1 GTPase- and sphingolipid-dependent manner. The sphingolipids, but not Bud1, also contributed to barrier formation in the outer membrane of the dividing nucleus. Barrier-dependent confinement of ER stress into the mother cell promoted aging. Together, our data clarify the physical nature of lateral diffusion barriers in the ER and establish the role of such barriers in the asymmetric segregation of proteotoxic misfolded proteins during cell division and aging. DOI: http://dx.doi.org/10.7554/eLife.01883.001 PMID:24843009

  13. Modulation of sphingolipid metabolism with calorie restriction enhances insulin action in skeletal muscle

    PubMed Central

    Obanda, Diana N.; Yu, Yongmei; Wang, Zhong Q; Cefalu, William T.

    2015-01-01

    This study sought to investigate the effect of calorie restriction (CR) on skeletal muscle sphingolipid metabolism and its contribution to improved insulin action in rats after a 6 month feeding study. Twenty nine (29) male Fischer-344 rats were randomized to an ad libitum (AL) diet or 30% CR. Dietary intake, body weight, and insulin sensitivity were monitored. After 6 months, skeletal muscle (vastus lateralis) was obtained for insulin signaling and lipid profiling. Calorie restriction significantly decreased insulin and glucose levels and also altered the expression and activity of proteins involved in sphingolipid formation and metabolism. The quantities of ceramides significantly increased in CR animals (p<0.05; n=14–15), while ceramide metabolism products (i.e glycosphingolipids: hexosylceramides and lactosylceramides) significantly decreased (p<0.05; n=14–15). Ceramide phosphates, sphingomyelins, sphingosine and sphingosine phosphate were not significantly different between AL and CR groups (p=ns; n=14–15). Lactosylceramide quantities correlated significantly with surrogate markers of insulin resistance (HOMA-IR) (r=0.7, p<0.005). Products of ceramide metabolism (glycosphingolipids), known to interfere with insulin signaling at elevated levels were significantly reduced in the skeletal muscle of CR animals. The increase in insulin sensitivity is associated with glycosphingolipid levels. PMID:25771159

  14. Hepatic fatty acid uptake is regulated by the sphingolipid acyl chain length

    PubMed Central

    Park, Woo-Jae; Park, Joo-Won; Merrill, Alfred H.; Storch, Judith; Pewzner-Jung, Yael; Futerman, Anthony H.

    2015-01-01

    Ceramide synthase 2 (CerS2) null mice cannot synthesize very-long acyl chain (C22-C24) ceramides resulting in significant alterations in the acyl chain composition of sphingolipids. We now demonstrate that hepatic triacylglycerol (TG) levels are reduced in liver but not in adipose tissue or skeletal muscle in the CerS2 null mouse, both before and after feeding with a high fat diet (HFD), where no weight gain was observed and large hepatic nodules appeared. Uptake of both BODIPY-palmitate and [3H]-palmitate were also abrogated in hepatocytes and liver. The role of a number of key proteins involved in fatty acid uptake was examined, including FATP5, CD36/FAT, FABPpm and cytoplasmic FABP1. Levels of FATP5 and FABP1 were decreased in CerS2 null mouse liver, whereas CD36/FAT levels were significantly elevated and CD36/FAT was also mislocalized upon insulin treatment. Moreover, treatment of hepatocytes with C22-C24-ceramides down-regulated CD36/FAT levels. Infection of CerS2 null mice with recombinant adeno-associated virus (rAAV)-CerS2 restored normal TG levels and corrected the mislocalization of CD36/FAT, but had no effect on the intracellular localization or levels of FATP5 or FABP1. Together, these results demonstrate that hepatic fatty acid uptake via CD36/FAT can be regulated by altering the acyl chain composition of sphingolipids. PMID:25241943

  15. The ATP-binding cassette transporter-2 (ABCA2) regulates esterification of plasma membrane cholesterol by modulation of sphingolipid metabolism.

    PubMed

    Davis, Warren

    2014-01-01

    The ATP-binding cassette transporters are a large family (~48 genes divided into seven families A-G) of proteins that utilize the energy of ATP-hydrolysis to pump substrates across lipid bilayers against a concentration gradient. The ABC "A" subfamily is comprised of 13 members and transport sterols, phospholipids and bile acids. ABCA2 is the most abundant ABC transporter in human and rodent brain with highest expression in oligodendrocytes, although it is also expressed in neurons. Several groups have studied a possible connection between ABCA2 and Alzheimer's disease as well as early atherosclerosis. ABCA2 expression levels have been associated with changes in cholesterol and sphingolipid metabolism. In this paper, we hypothesized that ABCA2 expression level may regulate esterification of plasma membrane-derived cholesterol by modulation of sphingolipid metabolism. ABCA2 overexpression in N2a neuroblastoma cells was associated with an altered bilayer distribution of the sphingolipid ceramide that inhibited acylCoA:cholesterol acyltransferase (ACAT) activity and cholesterol esterification. In contrast, depletion of endogenous ABCA2 in the rat schwannoma cell line D6P2T increased esterification of plasma membrane cholesterol following treatment with exogenous bacterial sphingomyelinase. These findings suggest that control of ABCA2 expression level may be a key locus of regulation for esterification of plasma membrane-derived cholesterol through modulation of sphingolipid metabolism.

  16. Enhanced apoptotic cancer cell killing after Foscan photodynamic therapy combined with fenretinide via de novo sphingolipid biosynthesis pathway.

    PubMed

    Boppana, Nithin B; DeLor, Jeremy S; Van Buren, Eric; Bielawska, Alicja; Bielawski, Jacek; Pierce, Jason S; Korbelik, Mladen; Separovic, Duska

    2016-06-01

    We and others have shown that stresses, including photodynamic therapy (PDT), can disrupt the de novo sphingolipid biosynthesis pathway, leading to changes in the levels of sphingolipids, and subsequently, modulation of cell death. The de novo sphingolipid biosynthesis pathway includes a ceramide synthase-dependent reaction, giving rise to dihydroceramide, which is then converted in a desaturase-dependent reaction to ceramide. In this study we tested the hypothesis that combining Foscan-mediated PDT with desaturase inhibitor fenretinide (HPR) enhances cancer cell killing. We discovered that by subjecting SCC19 cells, a human head and neck squamous cell carcinoma cell line, to PDT+HPR resulted in enhanced accumulation of C16-dihydroceramide, not ceramide. Concomitantly, mitochondrial depolarization was enhanced by the combined treatment. Enhanced activation of caspase-3 after PDT+HPR was inhibited by FB. Enhanced clonogenic cell death after the combination was sensitive to FB, as well as Bcl2- and caspase inhibitors. Treatment of mouse SCCVII squamous cell carcinoma tumors with PDT+HPR resulted in improved long-term tumor cures. Overall, our data showed that combining PDT with HPR enhanced apoptotic cancer cell killing and antitumor efficacy of PDT. The data suggest the involvement of the de novo sphingolipid biosynthesis pathway in enhanced apoptotic cell killing after PDT+HPR, and identify the combination as a novel more effective anticancer treatment than either treatment alone. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Arabidopsis mutants in sphingolipid synthesis as tools to understand the structure and function of membrane microdomains in plasmodesmata

    PubMed Central

    González-Solís, Ariadna; Cano-Ramírez, Dora L.; Morales-Cedillo, Francisco; Tapia de Aquino, Cinthya; Gavilanes-Ruiz, Marina

    2013-01-01

    Plasmodesmata—intercellular channels that communicate adjacent cells—possess complex membranous structures. Recent evidences indicate that plasmodesmata contain membrane microdomains. In order to understand how these submembrane regions collaborate to plasmodesmata function, it is necessary to characterize their size, composition and dynamics. An approach that can shed light on these microdomain features is based on the use of Arabidopsis mutants in sphingolipid synthesis. Sphingolipids are canonical components of microdomains together with sterols and some glycerolipids. Moreover, sphingolipids are transducers in pathways that display programmed cell death as a defense mechanism against pathogens. The study of Arabidopsis mutants would allow determining which structural features of the sphingolipids are important for the formation and stability of microdomains, and if defense signaling networks using sphingoid bases as second messengers are associated to plasmodesmata operation. Such studies need to be complemented by analysis of the ultrastructure and the use of protein probes for plasmodesmata microdomains and may constitute a very valuable source of information to analyze these membrane structures. PMID:24478783

  18. Sphingolipids regulate [Mg2+]o uptake and [Mg2+]i content in vascular smooth muscle cells: potential mechanisms and importance to membrane transport of Mg2+.

    PubMed

    Zheng, Tao; Li, Wenyan; Altura, Bella T; Shah, Nilank C; Altura, Burton M

    2011-02-01

    Sphingolipids have a variety of important signaling roles in mammalian cells. We tested the hypothesis that certain sphingolipids and neutral sphingomyelinase (N-SMase) can regulate intracellular free magnesium ions ([Mg2+]i) in vascular smooth muscle (VSM) cells. Herein, we show that several sphingolipids, including C2-ceramide, C8-ceramide, C16-ceramide, and sphingosine, as well as N-SMase, have potent and direct effects on content and mobilization of [Mg2+]i in primary cultured rat aortic smooth muscle cells. All of these sphingolipid molecules increase, rapidly, [Mg2+]i in these vascular cells in a concentration-dependent manner. The increments of [Mg2+]i, induced by these agents, are derived from influx of extracellular Mg2+ and are extracellular Ca2+ concentration-dependent. Phospholipase C and Ca2+/calmodulin/Ca2+-ATPase activity appear to be important in the sphingolipid-induced rises of [Mg2+]i. Activation of certain PKC isozymes may also be required for sphingolipid-induced rises in [Mg2+]i. These novel results suggest that sphingolipids may be homeostatic regulators of extracellular Mg2+ concentration influx (and transport) and [Mg2+]i content in vascular muscle cells.

  19. Unravelling the interplay of sphingolipids and TGF-β signaling in the human corneal stroma.

    PubMed

    Nicholas, Sarah E; Rowsey, Tyler G; Priyadarsini, Shrestha; Mandal, Nawajes A; Karamichos, Dimitrios

    2017-01-01

    To delineate the role of Sphingolipids (SPLs) in the human cornea and their cross-talks with transforming growth factor beta (TGF-β) in order to develop novel, non-invasive therapies. Human corneal fibroblasts (HCFs) were harvested from healthy donors, stimulated with Vitamin C to promote extracellular matrix assembly, treated with exogenous sphingosine-1-phosphate (S1P) or sphingosine kinase inhibitor 2 (SPHK I2) and isolated after 4 weeks for further analysis. Data showed that S1P led to a significant decrease in cellular migration where SPHK I2 just delayed it for 24h. Significant modulation of the sphingolipid pathway was also noted. Sphingosine kinase-1 (SphK1) was significantly downregulated upon exogenous stimulation with S1P at a concentration of 5μM and Sphingosine kinase-2 (SphK2) was also significantly downregulated at concentrations of 0.01μM, 0.1μM, and 5μM; whereas no effects were observed upon stimulation with SPHK I2. S1PR3 was significantly downregulated by 0.1μM and 5μM S1P and upregulated by 5μM and 10μM SPHK I2. Furthermore, both S1P and SPHK I2 regulated corneal fibrosis markers such as alpha-smooth muscle actin, collagen I, III, and V. We also investigated the interplay between two TGF-β isoforms and S1P/SPHK I2 treatments and found that TGF-β1 and TGF-β3 were both significantly upregulated with the 0.1μM S1P but were significantly downregulated with the 5μM S1P concentration. When TGF-β1 was compared directly to TGF-β3 expression, we observed that TGF-β3 was significantly downregulated compared to TGF-β1 in the 5μM concentration of S1P. No changes were observed upon SPHK I2 treatment. Our study delineates the role of sphingolipids in the human cornea and highlights their different activities based on the cell/tissue type.

  20. Sphingolipid biosynthesis upregulation by TOR complex 2-Ypk1 signaling during yeast adaptive response to acetic acid stress.

    PubMed

    Guerreiro, Joana F; Muir, Alexander; Ramachandran, Subramaniam; Thorner, Jeremy; Sá-Correia, Isabel

    2016-12-01

    Acetic acid-induced inhibition of yeast growth and metabolism limits the productivity of industrial fermentation processes, especially when lignocellulosic hydrolysates are used as feedstock in industrial biotechnology. Tolerance to acetic acid of food spoilage yeasts is also a problem in the preservation of acidic foods and beverages. Thus understanding the molecular mechanisms underlying adaptation and tolerance to acetic acid stress is increasingly important in industrial biotechnology and the food industry. Prior genetic screens for Saccharomyces cerevisiae mutants with increased sensitivity to acetic acid identified loss-of-function mutations in the YPK1 gene, which encodes a protein kinase activated by the target of rapamycin (TOR) complex 2 (TORC2). We show in the present study by several independent criteria that TORC2-Ypk1 signaling is stimulated in response to acetic acid stress. Moreover, we demonstrate that TORC2-mediated Ypk1 phosphorylation and activation is necessary for acetic acid tolerance, and occurs independently of Hrk1, a protein kinase previously implicated in the cellular response to acetic acid. In addition, we show that TORC2-Ypk1-mediated activation of l-serine:palmitoyl-CoA acyltransferase, the enzyme complex that catalyzes the first committed step of sphingolipid biosynthesis, is required for acetic acid tolerance. Furthermore, analysis of the sphingolipid pathway using inhibitors and mutants indicates that it is production of certain complex sphingolipids that contributes to conferring acetic acid tolerance. Consistent with that conclusion, promoting sphingolipid synthesis by adding exogenous long-chain base precursor phytosphingosine to the growth medium enhanced acetic acid tolerance. Thus appropriate modulation of the TORC2-Ypk1-sphingolipid axis in industrial yeast strains may have utility in improving fermentations of acetic acid-containing feedstocks. © 2016 The Author(s); published by Portland Press Limited on behalf of the

  1. Sphingolipid biosynthesis upregulation by TOR Complex 2-Ypk1 signaling during yeast adaptive response to acetic acid stress

    PubMed Central

    Guerreiro, Joana F.; Muir, Alexander; Ramachandran, Subramaniam; Thorner, Jeremy; Sá-Correia, Isabel

    2016-01-01

    Acetic acid-induced inhibition of yeast growth and metabolism limits the productivity of industrial fermentation processes, especially when lignocellulosic hydrolysates are used as feedstock in industrial biotechnology. Tolerance to acetic acid of food spoilage yeasts is also a problem in the preservation of acidic foods and beverages. Thus, understanding the molecular mechanisms underlying adaptation and tolerance to acetic acid stress is increasingly important in industrial biotechnology and the food industry. Prior genetic screens for S. cerevisiae mutants with increased sensitivity to acetic acid identified loss-of-function mutations in the YPK1 gene, which encodes a protein kinase activated by the Target of Rapamycin (TOR) Complex 2 (TORC2). We show here by several independent criteria that TORC2-Ypk1 signaling is stimulated in response to acetic acid stress. Moreover, we demonstrate that TORC2-mediated Ypk1 phosphorylation and activation is necessary for acetic acid tolerance, and occurs independently of Hrk1, a protein kinase previously implicated in the cellular response to acetic acid. In addition, we show that TORC2-Ypk1-mediated activation of L-serine: palmitoyl-CoA acyltransferase, the enzyme complex that catalyzes the first committed step of sphingolipid biosynthesis, is required for acetic acid tolerance. Furthermore, analysis of the sphingolipid pathway using inhibitors and mutants indicates that it is production of certain complex sphingolipids that contributes to conferring acetic acid tolerance. Consistent with that conclusion, promoting sphingolipid synthesis by adding exogenous long-chain base precursor phytosphingosine to the growth medium enhanced acetic acid tolerance. Thus, appropriate modulation of the TORC2-Ypk1-sphingolipid axis in industrial yeast strains may have utility in improving fermentations of acetic acid-containing feedstocks. PMID:27671892

  2. Sphingolipids and cancer: ceramide and sphingosine-1-phosphate in the regulation of cell death and drug resistance

    PubMed Central

    Ponnusamy, Suriyan; Meyers-Needham, Marisa; Senkal, Can E; Saddoughi, Sahar A; Sentelle, David; Selvam, Shanmugam Panneer; Salas, Arelis; Ogretmen, Besim

    2011-01-01

    Sphingolipids have emerged as bioeffector molecules, controlling various aspects of cell growth and proliferation in cancer, which is becoming the deadliest disease in the world. These lipid molecules have also been implicated in the mechanism of action of cancer chemotherapeutics. Ceramide, the central molecule of sphingolipid metabolism, generally mediates antiproliferative responses, such as cell growth inhibition, apoptosis induction, senescence modulation, endoplasmic reticulum stress responses and/or autophagy. Interestingly, recent studies suggest de novo-generated ceramides may have distinct and opposing roles in the promotion/suppression of tumors, and that these activities are based on their fatty acid chain lengths, subcellular localization and/or direct downstream targets. For example, in head and neck cancer cells, ceramide synthase 6/C16-ceramide addiction was revealed, and this was associated with increased tumor growth, whereas downregulation of its synthesis resulted in ER stress-induced apoptosis. By contrast, ceramide synthase 1-generated C18-ceramide has been shown to suppress tumor growth in various cancer models, both in situ and in vivo. In addition, ceramide metabolism to generate sphingosine-1-phosphate (S1P) by sphingosine kinases 1 and 2 mediates, with or without the involvement of G-protein-coupled S1P receptor signaling, prosurvival, angiogenesis, metastasis and/or resistance to drug-induced apoptosis. Importantly, recent findings regarding the mechanisms by which sphingolipid metabolism and signaling regulate tumor growth and progression, such as identifying direct intracellular protein targets of sphingolipids, have been key for the development of new chemotherapeutic strategies. Thus, in this article, we will present conclusions of recent studies that describe opposing roles of de novo-generated ceramides by ceramide synthases and/or S1P in the regulation of cancer pathogenesis, as well as the development of sphingolipid-based cancer

  3. Nitric oxide-sphingolipid interplays in plant signalling: a new enigma from the Sphinx?

    PubMed

    Guillas, Isabelle; Puyaubert, Juliette; Baudouin, Emmanuel

    2013-09-12

    Nitric oxide (NO) emerged as one of the major signaling molecules operating during plant development and plant responses to its environment. Beyond the identification of the direct molecular targets of NO, a series of studies considered its interplay with other actors of signal transduction and the integration of NO into complex signaling networks. Beside the close relationships between NO and calcium or phosphatidic acid signaling pathways that are now well-established, recent reports paved the way for interplays between NO and sphingolipids (SLs). This mini-review summarizes our current knowledge of the influence NO and SLs might exert on each other in plant physiology. Based on comparisons with examples from the animal field, it further indicates that, although SL-NO interplays are common features in signaling networks of eukaryotic cells, the underlying mechanisms and molecular targets significantly differ.

  4. Sphingolipids are required for efficient triacylglycerol loss in conjugated linoleic Acid treated adipocytes.

    PubMed

    Wang, Wei; Fromm, Michael

    2015-01-01

    Conjugated linoleic acid (CLA) reduces adiposity in human and mouse adipocytes. This outcome is achieved through a variety of biological responses including increased energy expenditure and fatty acid oxidation, increased inflammation, repression of fatty acid biosynthesis, attenuated glucose transport, and apoptosis. In the current study, profiling of 261 metabolites was conducted to gain new insights into the biological pathways responding to CLA in 3T3-L1 adipocytes. Sphinganine and sphingosine levels were observed to be highly elevated in CLA treated adipocytes. Exogenous chemicals that increased endogenous ceramide levels decreased lipid levels in adipocytes, and activated AMP-activated protein kinase (AMPK) as well as NF-κB, both of which are typically activated in CLA treated adipocytes. Concurrent inhibition of ceramide de novo biosynthesis and recycling from existing sphingolipid pools attenuated the lipid lowering effect normally associated with responses to CLA, implicating ceramides as an important component of the lipid lowering response in CLA treated adipocytes.

  5. The Sphingolipid Biosynthetic Pathway Is a Potential Target for Chemotherapy against Chagas Disease

    PubMed Central

    Koeller, Carolina Macedo; Heise, Norton

    2011-01-01

    The protozoan parasite Trypanosoma cruzi is the causative agent of human Chagas disease, for which there currently is no cure. The life cycle of T. cruzi is complex, including an extracellular phase in the triatomine insect vector and an obligatory intracellular stage inside the vertebrate host. These phases depend on a variety of surface glycosylphosphatidylinositol-(GPI-) anchored glycoconjugates that are synthesized by the parasite. Therefore, the surface expression of GPI-anchored components and the biosynthetic pathways of GPI anchors are attractive targets for new therapies for Chagas disease. We identified new drug targets for chemotherapy by taking the available genome sequence information and searching for differences in the sphingolipid biosynthetic pathways (SBPs) of mammals and T. cruzi. In this paper, we discuss the major steps of the SBP in mammals, yeast and T. cruzi, focusing on the IPC synthase and ceramide remodeling of T. cruzi as potential therapeutic targets for Chagas disease. PMID:21603271

  6. Sphingolipids Are Required for Efficient Triacylglycerol Loss in Conjugated Linoleic Acid Treated Adipocytes

    PubMed Central

    Wang, Wei; Fromm, Michael

    2015-01-01

    Conjugated linoleic acid (CLA) reduces adiposity in human and mouse adipocytes. This outcome is achieved through a variety of biological responses including increased energy expenditure and fatty acid oxidation, increased inflammation, repression of fatty acid biosynthesis, attenuated glucose transport, and apoptosis. In the current study, profiling of 261 metabolites was conducted to gain new insights into the biological pathways responding to CLA in 3T3-L1 adipocytes. Sphinganine and sphingosine levels were observed to be highly elevated in CLA treated adipocytes. Exogenous chemicals that increased endogenous ceramide levels decreased lipid levels in adipocytes, and activated AMP-activated protein kinase (AMPK) as well as NF-κB, both of which are typically activated in CLA treated adipocytes. Concurrent inhibition of ceramide de novo biosynthesis and recycling from existing sphingolipid pools attenuated the lipid lowering effect normally associated with responses to CLA, implicating ceramides as an important component of the lipid lowering response in CLA treated adipocytes. PMID:25906159

  7. Serum profiling of healthy aging identifies phospho- and sphingolipid species as markers of human longevity.

    PubMed

    Montoliu, Ivan; Scherer, Max; Beguelin, Fiona; DaSilva, Laeticia; Mari, Daniela; Salvioli, Stefano; Martin, Francois-Pierre J; Capri, Miriam; Bucci, Laura; Ostan, Rita; Garagnani, Paolo; Monti, Daniela; Biagi, Elena; Brigidi, Patrizia; Kussmann, Martin; Rezzi, Serge; Franceschi, Claudio; Collino, Sebastiano

    2014-01-01

    As centenarians well represent the model of healthy aging, there are many important implications in revealing the underlying molecular mechanisms behind such successful aging. By combining NMR metabonomics and shot-gun lipidomics in serum we analyzed metabolome and lipidome composition of a group of centenarians with respect to elderly individuals. Specifically, NMR metabonomics profiling of serum revealed that centenarians are characterized by a metabolic phenotype distinct from that of elderly subjects, in particular regarding amino acids and lipid species. Shot- gun lipidomics approach displays unique changes in lipids biosynthesis in centenarians, with 41 differently abundant lipid species with respect to elderly subjects. These findings reveal phospho/sphingolipids as putative markers and biological modulators of healthy aging, in humans. Considering the particular actions of these metabolites, these data are suggestive of a better counteractive antioxidant capacity and a well-developed membrane lipid remodelling process in the healthy aging phenotype.

  8. “Evolving concepts in cancer therapy through targeting sphingolipid metabolism”

    PubMed Central

    Truman, Jean-Philip; García-Barros, Mónica; Obeid, Lina M.

    2014-01-01

    Traditional methods of cancer treatment are limited in their efficacy due to both inherent and acquired factors. Many different studies have shown that the generation of ceramide in response to cytotoxic therapy is generally an important step leading to cell death. Cancer cells employ different methods to both limit ceramide generation and to remove ceramide in order to become resistant to treatment. Furthermore, sphingosine kinase activity, which phosphorylates sphingosine the product of ceramide hydrolysis, has been linked to multidrug resistance, and can act as a strong survival factor. This review will examine several of the most frequently used cancer therapies and their effect on both ceramide generation and the mechanisms employed to remove it. The development and use of inhibitors of sphingosine kinase will be focused upon as an example of how targeting sphingolipid metabolism may provide an effective means to improve treatment response rates and reduce associated treatment toxicity. PMID:24384461

  9. Long-term food restriction prevents aging-associated sphingolipid turnover dysregulation in the brain.

    PubMed

    Babenko, Nataliya A; Shakhova, Elena G

    2014-01-01

    Abnormalities of sphingolipid turnover in the brain during normal aging and age-related neurological disorders were associated with the neurons loss and cognitive malfunction. Calorie restriction (CR) prevented age-related deficits in hippocampal long-term potentiation and improved cognitive function at old age. In the paper we investigated the ceramide and sphingomyelin (SM) levels in the brain regions, which are critical for learning and memory of 3- and 24-month-old rats, as well as the correction of sphingolipid turnover in the brain of old rats, by means of the CR diet and modulators of SM turnover. Using the [methyl-(14)C-choline]SM, the neutral, but not the acid SMase activity has been observed to increase in both the hippocampus and brain cortex of 24-month-old rats with respect to 3-month-old animals. Age-dependent changes of neutral SMase activities were associated with ceramide accumulation and SM level drop in the brain structures studied. Treatment of the rats with the CR diet or N-acetylcysteine (NAC) or α-tocopherol acetate, but not an inhibitor of acid SMase imipramine, reduced the ceramide content and neutral SMase activity in the hippocampus of 24-month-old animals with respect to control rats of the same age. These results suggest that redox-sensitive neutral SMase plays important role in SM turnover dysregulation in both the hippocampus and neocortex at old age and that the CR diet can prevent the age-dependent accumulation of ceramide mainly via neutral SMase targeting. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  10. Neurochemical Metabolomics Reveals Disruption to Sphingolipid Metabolism Following Chronic Haloperidol Administration.

    PubMed

    McClay, Joseph L; Vunck, Sarah A; Batman, Angela M; Crowley, James J; Vann, Robert E; Beardsley, Patrick M; van den Oord, Edwin J

    2015-09-01

    Haloperidol is an effective antipsychotic drug for treatment of schizophrenia, but prolonged use can lead to debilitating side effects. To better understand the effects of long-term administration, we measured global metabolic changes in mouse brain following 3 mg/kg/day haloperidol for 28 days. These conditions lead to movement-related side effects in mice akin to those observed in patients after prolonged use. Brain tissue was collected following microwave tissue fixation to arrest metabolism and extracted metabolites were assessed using both liquid and gas chromatography mass spectrometry (MS). Over 300 unique compounds were identified across MS platforms. Haloperidol was found to be present in all test samples and not in controls, indicating experimental validity. Twenty-one compounds differed significantly between test and control groups at the p < 0.05 level. Top compounds were robust to analytical method, also being identified via partial least squares discriminant analysis. Four compounds (sphinganine, N-acetylornithine, leucine and adenosine diphosphate) survived correction for multiple testing in a non-parametric analysis using false discovery rate threshold < 0.1. Pathway analysis of nominally significant compounds (p < 0.05) revealed significant findings for sphingolipid metabolism (p = 0.015) and protein biosynthesis (p = 0.024). Altered sphingolipid metabolism is suggestive of disruptions to myelin. This interpretation is supported by our observation of elevated N-acetyl-aspartyl-glutamate in the haloperidol-treated mice (p = 0.004), a marker previously associated with demyelination. This study further demonstrates the utility of murine neurochemical metabolomics as a method to advance understanding of CNS drug effects.

  11. Neurochemical metabolomics reveals disruption to sphingolipid metabolism following chronic haloperidol administration

    PubMed Central

    McClay, Joseph L.; Vunck, Sarah A.; Batman, Angela M.; Crowley, James J.; Vann, Robert E.; Beardsley, Patrick M.; van den Oord, Edwin J.

    2015-01-01

    Haloperidol is an effective antipsychotic drug for treatment of schizophrenia, but prolonged use can lead to debilitating side effects. To better understand the effects of long-term administration, we measured global metabolic changes in mouse brain following 3 mg/kg/day haloperidol for 28 days. These conditions lead to movement-related side effects in mice akin to those observed in patients after prolonged use. Brain tissue was collected following microwave tissue fixation to arrest metabolism and extracted metabolites were assessed using both liquid and gas chromatography mass spectrometry (MS). Over 300 unique compounds were identified across MS platforms. Haloperidol was found to be present in all test samples and not in controls, indicating experimental validity. Twenty-one compounds differed significantly between test and control groups at the p < 0.05 level. Top compounds were robust to analytical method, also being identified via partial least squares discriminant analysis. Four compounds (sphinganine, N-acetylornithine, leucine and adenosine diphosphate) survived correction for multiple testing in a non-parametric analysis using false discovery rate threshold < 0.1. Pathway analysis of nominally significant compounds (p < 0.05) revealed significant findings for sphingolipid metabolism (p = 0.02) and protein biosynthesis (p = 0.03). Altered sphingolipid metabolism is suggestive of disruptions to myelin. This interpretation is supported by our observation of elevated N-acetylaspartylglutamate in the haloperidol-treated mice (p = 0.004), a marker previously associated with demyelination. This study further demonstrates the utility of murine neurochemical metabolomics as a method to advance understanding of CNS drug effects. PMID:25850894

  12. A Genome-Wide Enhancer Screen Implicates Sphingolipid Composition in Vacuolar ATPase Function in Saccharomyces cerevisiae

    PubMed Central

    Finnigan, Gregory C.; Ryan, Margret; Stevens, Tom H.

    2011-01-01

    The function of the vacuolar H+-ATPase (V-ATPase) enzyme complex is to acidify organelles; this process is critical for a variety of cellular processes and has implications in human disease. There are five accessory proteins that assist in assembly of the membrane portion of the complex, the V0 domain. To identify additional elements that affect V-ATPase assembly, trafficking, or enzyme activity, we performed a genome-wide enhancer screen in the budding yeast Saccharomyces cerevisiae with two mutant assembly factor alleles, VMA21 with a dysfunctional ER retrieval motif (vma21QQ) and vma21QQ in combination with voa1Δ, a nonessential assembly factor. These alleles serve as sensitized genetic backgrounds that have reduced V-ATPase enzyme activity. Genes were identified from a variety of cellular pathways including a large number of trafficking-related components; we characterized two redundant gene pairs, HPH1/HPH2 and ORM1/ORM2. Both sets demonstrated synthetic growth defects in combination with the vma21QQ allele. A loss of either the HPH or ORM gene pairs alone did not result in a decrease in vacuolar acidification or defects in V-ATPase assembly. While the Hph proteins are not required for V-ATPase function, Orm1p and Orm2p are required for full V-ATPase enzyme function. Consistent with the documented role of the Orm proteins in sphingolipid regulation, we have found that inhibition of sphingolipid synthesis alleviates Orm-related growth defects. PMID:21196517

  13. Disruption of sphingolipid metabolism augments ceramide-induced autophagy in preeclampsia

    PubMed Central

    Melland-Smith, Megan; Ermini, Leonardo; Chauvin, Sarah; Craig-Barnes, Hayley; Tagliaferro, Andrea; Todros, Tullia; Post, Martin; Caniggia, Isabella

    2015-01-01

    Bioactive sphingolipids including ceramides are involved in a variety of pathophysiological processes by regulating cell death and survival. The objective of the current study was to examine ceramide metabolism in preeclampsia, a serious disorder of pregnancy characterized by oxidative stress, and increased trophoblast cell death and autophagy. Maternal circulating and placental ceramide levels quantified by tandem mass spectrometry were elevated in pregnancies complicated by preeclampsia. Placental ceramides were elevated due to greater de novo synthesis via high serine palmitoyltransferase activity and reduced lysosomal breakdown via diminished ASAH1 expression caused by TGFB3-induced E2F4 transcriptional repression. SMPD1 activity was reduced; hence, sphingomyelin degradation by SMPD1 did not contribute to elevated ceramide levels in preeclampsia. Oxidative stress triggered similar changes in ceramide levels and acid hydrolase expression in villous explants and trophoblast cells. MALDI-imaging mass spectrometry localized the ceramide increases to the trophophoblast layers and syncytial knots of placentae from pregnancies complicated by preeclampsia. ASAH1 inhibition or ceramide treatment induced autophagy in human trophoblast cells via a shift of the BOK-MCL1 rheostat toward prodeath BOK. Pharmacological inhibition of ASAH1 activity in pregnant mice resulted in increased placental ceramide content, abnormal placentation, reduced fetal growth, and increased autophagy via a similar shift in the BOK-MCL1 system. Our results reveal that oxidative stress-induced reduction of lysosomal hydrolase activities in combination with elevated de novo synthesis leads to ceramide overload, resulting in increased trophoblast cell autophagy, and typifies preeclampsia as a sphingolipid storage disorder. PMID:25853898

  14. A profile of sphingolipids and related compounds tentatively identified in yak milk.

    PubMed

    Qu, S; Barrett-Wilt, G; Fonseca, L M; Rankin, S A

    2016-07-01

    This work characterized a fraction of constituents in yak milk within the realm of approximately 1,000 to 3,000 Da using matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry. Eleven samples of yak milk powder from the Sichuan province of China were received by the Department of Food Science, University of Wisconsin-Madison, and stored at room temperature until analysis. Sample preparation involved delipidation and deproteinization of yak milk samples and cold ethanol precipitation. Subsequently, MALDI time-of-flight mass spectrometry was performed in positive ion, reflector mode (AB Sciex TOF/TOF 4800 MALDI; AB Sciex, Foster City, CA). The instrument was first calibrated with the manufacturer's 6-peptide mixture, and each spectrum was internally calibrated using the accurate mass of ACTH Fragment 18-39 standard peptide (protonated mass at m/z 2464.199) present in each sample. Laser power was adjusted for the calibration standards and for each sample so that the signal obtained for the most-abundant ion in each spectrum could be maximized, or kept below ~2×10(4) to preserve spectral quality. Structure and name based on mass were matched using the Metlin metabolite database (https://metlin.scripps.edu/index.php). Results of the current work for yak milk powder showed a large variety of sphingolipid structures with clusters around 1,200, 1,600, and 2,000 Da. The profiling matched several glycosphingolipids, such as gangliosides GA1, GD1a, GD1b, GD3, GM1, GM2, GM3, and GT2 and several other unique moieties, including deaminated neuraminic acid (KDN) oligosaccharides, and fucose containing gangliosides. Matrix preparation and MALDI time-of-flight parameters were important factors established in this work to allow high resolution profiling of complex sphingolipids in yak powder milk. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  15. TNF-α- and tumor-induced skeletal muscle atrophy involves sphingolipid metabolism.

    PubMed

    De Larichaudy, Joffrey; Zufferli, Alessandra; Serra, Filippo; Isidori, Andrea M; Naro, Fabio; Dessalle, Kevin; Desgeorges, Marine; Piraud, Monique; Cheillan, David; Vidal, Hubert; Lefai, Etienne; Némoz, Georges

    2012-01-18

    Muscle atrophy associated with various pathophysiological conditions represents a major health problem, because of its contribution to the deterioration of patient status and its effect on mortality. Although the involvement of pro-inflammatory cytokines in this process is well recognized, the role of sphingolipid metabolism alterations induced by the cytokines has received little attention. We addressed this question both in vitro using differentiated myotubes treated with TNF-α, and in vivo in a murine model of tumor-induced cachexia. Myotube atrophy induced by TNF-α was accompanied by a substantial increase in cell ceramide levels, and could be mimicked by the addition of exogenous ceramides. It could be prevented by the addition of ceramide-synthesis inhibitors that targeted either the de novo pathway (myriocin), or the sphingomyelinases (GW4869 and 3-O-methylsphingomyelin). In the presence of TNF-α, ceramide-synthesis inhibitors significantly increased protein synthesis and decreased proteolysis. In parallel, they lowered the expression of both the Atrogin-1 and LC3b genes, involved in muscle protein degradation by proteasome and in autophagic proteolysis, respectively, and increased the proportion of inactive, phosphorylated Foxo3 transcription factor. Furthermore, these inhibitors increased the expression and/or phosphorylation levels of key factors regulating protein metabolism, including phospholipase D, an activator of mammalian target of rapamycin (mTOR), and the mTOR substrates S6K1 and Akt. In vivo, C26 carcinoma implantation induced a substantial increase in muscle ceramide, together with drastic muscle atrophy. Treatment of the animals with myriocin reduced the expression of the atrogenes Foxo3 and Atrogin-1, and partially protected muscle tissue from atrophy. Ceramide accumulation induced by TNF-α or tumor development participates in the mechanism of muscle-cell atrophy, and sphingolipid metabolism is a logical target for pharmacological or

  16. TNF-α- and tumor-induced skeletal muscle atrophy involves sphingolipid metabolism

    PubMed Central

    2012-01-01

    Background Muscle atrophy associated with various pathophysiological conditions represents a major health problem, because of its contribution to the deterioration of patient status and its effect on mortality. Although the involvement of pro-inflammatory cytokines in this process is well recognized, the role of sphingolipid metabolism alterations induced by the cytokines has received little attention. Results We addressed this question both in vitro using differentiated myotubes treated with TNF-α, and in vivo in a murine model of tumor-induced cachexia. Myotube atrophy induced by TNF-α was accompanied by a substantial increase in cell ceramide levels, and could be mimicked by the addition of exogenous ceramides. It could be prevented by the addition of ceramide-synthesis inhibitors that targeted either the de novo pathway (myriocin), or the sphingomyelinases (GW4869 and 3-O-methylsphingomyelin). In the presence of TNF-α, ceramide-synthesis inhibitors significantly increased protein synthesis and decreased proteolysis. In parallel, they lowered the expression of both the Atrogin-1 and LC3b genes, involved in muscle protein degradation by proteasome and in autophagic proteolysis, respectively, and increased the proportion of inactive, phosphorylated Foxo3 transcription factor. Furthermore, these inhibitors increased the expression and/or phosphorylation levels of key factors regulating protein metabolism, including phospholipase D, an activator of mammalian target of rapamycin (mTOR), and the mTOR substrates S6K1 and Akt. In vivo, C26 carcinoma implantation induced a substantial increase in muscle ceramide, together with drastic muscle atrophy. Treatment of the animals with myriocin reduced the expression of the atrogenes Foxo3 and Atrogin-1, and partially protected muscle tissue from atrophy. Conclusions Ceramide accumulation induced by TNF-α or tumor development participates in the mechanism of muscle-cell atrophy, and sphingolipid metabolism is a logical

  17. Simultaneous quantitative analysis of bioactive sphingolipids by high-performance liquid chromatography-tandem mass spectrometry.

    PubMed

    Bielawski, Jacek; Szulc, Zdzislaw M; Hannun, Yusuf A; Bielawska, Alicja

    2006-06-01

    There has been a recent explosion in research concerning novel bioactive sphingolipids (SPLs) such as ceramide (Cer), sphingosine (Sph) and sphingosine 1-phosphate (Sph-1P) that necessitates development of accurate and user-friendly methodology for analyzing and quantitating the endogenous levels of these molecules. ESI/MS/MS methodology provides a universal tool used for detecting and monitoring changes in SPL levels and composition from biological materials. Simultaneous ESI/MS/MS analysis of sphingoid bases (SBs), sphingoid base 1-phosphates (SB-1Ps), Cers and sphingomyelins (SMs) is performed on a Thermo Finnigan TSQ 7000 triple quadrupole mass spectrometer operating in a multiple reaction monitoring (MRM) positive ionization mode. Biological materials (cells, tissues or physiological fluids) are fortified with internal standards (ISs), extracted into a one-phase neutral organic solvent system, and analyzed by a Surveyor/TSQ 7000 LC/MS system. Qualitative analysis of SPLs is performed by a Parent Ion scan of a common fragment ion characteristic for a particular class of SPLs. Quantitative analysis is based on calibration curves generated by spiking an artificial matrix with known amounts of target synthetic standards and an equal amount of IS. The calibration curves are constructed by plotting the peak area ratios of analyte to the respective IS against concentration using a linear regression model. This robust analytical procedure can determine the composition of endogenous sphingolipids (ESPLs) in varied biological materials and achieve a detection limit at 1 pmol or lower level. This and related methodology are already defining unexpected specialization and specificity in the metabolism and function of distinct subspecies of individual bioactive SPLs.

  18. Enrichment of hydroxylated C24- and C26-acyl-chain sphingolipids mediates PIN2 apical sorting at trans-Golgi network subdomains

    PubMed Central

    Wattelet-Boyer, Valérie; Brocard, Lysiane; Jonsson, Kristoffer; Esnay, Nicolas; Joubès, Jérôme; Domergue, Frédéric; Mongrand, Sébastien; Raikhel, Natasha; Bhalerao, Rishikesh P.; Moreau, Patrick; Boutté, Yohann

    2016-01-01

    The post-Golgi compartment trans-Golgi Network (TGN) is a central hub divided into multiple subdomains hosting distinct trafficking pathways, including polar delivery to apical membrane. Lipids such as sphingolipids and sterols have been implicated in polar trafficking from the TGN but the underlying mechanisms linking lipid composition to functional polar sorting at TGN subdomains remain unknown. Here we demonstrate that sphingolipids with α-hydroxylated acyl-chains of at least 24 carbon atoms are enriched in secretory vesicle subdomains of the TGN and are critical for de novo polar secretory sorting of the auxin carrier PIN2 to apical membrane of Arabidopsis root epithelial cells. We show that sphingolipid acyl-chain length influences the morphology and interconnections of TGN-associated secretory vesicles. Our results uncover that the sphingolipids acyl-chain length links lipid composition of TGN subdomains with polar secretory trafficking of PIN2 to apical membrane of polarized epithelial cells. PMID:27681606

  19. Dominant suppressor mutation bypasses the sphingolipid requirement for growth of Saccharomyces cells at low pH: role of the CWP2 gene.

    PubMed

    Skrzypek, M; Lester, R L; Spielmann, P; Zingg, N; Shelling, J; Dickson, R C

    2000-11-01

    Strains of Saccharomyces cerevisiae termed sphingolipid compensatory (SLC) do not grow at low pH when the cells lack sphingolipids. To begin to understand why sphingolipids are required for growth at low pH, we isolated derivatives of SLC strains, termed low pH resistant (LprR), carrying the LPR suppressor gene that allows growth at pH 4.1 when cells lack sphingolipids. Suppression is due to mutation of a single nuclear gene. The LPR suppressor gene functions, at least in part, by enhancing the ability of cells lacking sphingolipids to generate a net efflux of protons in suspension fluid with a pH range of 4.0-6.0. The LPR suppressor gene also enables cells lacking sphingolipids to maintain their intracellular pH near neutrality when the pH of the suspension fluid is low, unlike cells lacking the suppressor gene, which cannot maintain their intracellular pH in the face of a low external pH. These results demonstrate that some functions(s) of sphingolipids necessary for growth at low pH can be bypassed by a suppressor mutation. Attempts to clone the LPR suppressor gene were not successful, but they led to the isolation of the CWP2 gene, which encodes a major mannoprotein component of the outer cell wall. It was isolated because an increased copy number has the unusual property of increasing the frequency at which LprR strains arise. As we show here, part of the reason for this effect is that the CWP2 gene is essential for generating a net efflux of protons and for controlling intracellular pH in LprR strains that lack sphingolipids. These results suggest new cellular functions for the Cwp2 protein.

  20. Extensive sphingolipid depletion does not affect lipid raft integrity or lipid raft localization and efflux function of the ABC transporter MRP1

    PubMed Central

    Klappe, Karin; Dijkhuis, Anne-Jan; Hummel, Ina; vanDam, Annie; Ivanova, Pavlina T.; Milne, Stephen B.; Myers, David S.; Brown, H. Alex; Permentier, Hjalmar; Kok, Jan W.

    2013-01-01

    We show that highly efficient depletion of sphingolipids in two different cell lines does not abrogate the ability to isolate Lubrol-based DRMs (detergent-resistant membranes) or detergent-free lipid rafts from these cells. Compared with control, DRM/detergent-free lipid raft fractions contain equal amounts of protein, cholesterol and phospholipid, whereas the classical DRM/lipid raft markers Src, caveolin-1 and flotillin display the same gradient distribution. DRMs/detergent-free lipid rafts themselves are severely depleted of sphingolipids. The fatty acid profile of the remaining sphingolipids as well as that of the glycerophospholipids shows several differences compared with control, most prominently an increase in highly saturated C16 species. The glycerophospholipid headgroup composition is unchanged in sphingolipid-depleted cells and cell-derived detergent-free lipid rafts. Sphingolipid depletion does not alter the localization of MRP1 (multidrug-resistance-related protein 1) in DRMs/detergent-free lipid rafts or MRP1-mediated efflux of carboxyfluorescein. We conclude that extensive sphingolipid depletion does not affect lipid raft integrity in two cell lines and does not affect the function of the lipid-raft-associated protein MRP1. PMID:20604746

  1. Sphingolipids in marine microalgae: Development and application of a mass spectrometric method for global structural characterization of ceramides and glycosphingolipids in three major phyla.

    PubMed

    Li, Yanrong; Lou, Yamin; Mu, Tong; Ke, Aiying; Ran, Zhaoshou; Xu, Jilin; Chen, Juanjuan; Zhou, Chengxu; Yan, Xiaojun; Xu, Qingshan; Tan, Yinghong

    2017-09-15

    Sphingolipid compositions are crucial for the structural and physiological properties of microalgae membranes. In the present study, we developed a quadrupole time-of-flight (Q-TOF) mass spectrometric method based on MS(E) data collection for the identification of sphingolipids with high efficiency, selectivity, sensitivity and mass accuracy and applied this method for precise structural identification and quantitative profiling of ceramides and glycosphingolipids in total lipid extracts from 17 strains of microalgae, including 11 strains of diatom, 3 strains of dinoflagellate and 3 strains of haptophyta. Using this method, four species of sphingolipids including 27 ceramides, 13 monosaccharide ceramides, 18 disaccharide ceramides and 18 trisaccharide ceramides were identified. The compositions of sphingolipid-included glycosyl moieties, long chain bases and N-acyl chains showed a significant difference among different microalgae categories. Some long chain bases including d19:2, d19:3 and d19:4, glycosyl moieties including disaccharide and trisaccharide, and N-acyl chains such as 14:0, 14:1, 24:0, 24:1, h18:1, h19:1 and h22:0-2 can be chosen as the molecular signature for microalgae from three major phyla. This methodology will be useful for a wide range of physiological and pathological studies of sphingolipids. Furthermore, the diversity of sphingolipid structure could provide a new criterion for microalgae chemotaxonomy. Copyright © 2017. Published by Elsevier B.V.

  2. Orosomucoid Proteins Interact with the Small Subunit of Serine Palmitoyltransferase and Contribute to Sphingolipid Homeostasis and Stress Responses in Arabidopsis[OPEN

    PubMed Central

    Li, Jian; Yin, Jian; Rong, Chan; Li, Kai-En; Wu, Jian-Xin; Huang, Li-Qun; Zeng, Hong-Yun; Sahu, Sunil Kumar; Yao, Nan

    2016-01-01

    Serine palmitoyltransferase (SPT), a pyridoxyl-5′-phosphate-dependent enzyme, catalyzes the first and rate-limiting step in sphingolipid biosynthesis. In humans and yeast, orosomucoid proteins (ORMs) negatively regulate SPT and thus play an important role in maintaining sphingolipid levels. Despite the importance of sphingoid intermediates as bioactive molecules, the regulation of sphingolipid biosynthesis through SPT is not well understood in plants. Here, we identified and characterized the Arabidopsis thaliana ORMs, ORM1 and ORM2. Loss of function of both ORM1 and ORM2 (orm1 amiR-ORM2) stimulated de novo sphingolipid biosynthesis, leading to strong sphingolipid accumulation, especially of long-chain bases and ceramides. Yeast two-hybrid, bimolecular fluorescence complementation, and coimmunoprecipitation assays confirmed that ORM1 and ORM2 physically interact with the small subunit of SPT (ssSPT), indicating that ORMs inhibit ssSPT function. We found that orm1 amiR-ORM2 plants exhibited an early-senescence phenotype accompanied by H2O2 production at the cell wall and in mitochondria, active vesicular trafficking, and formation of cell wall appositions. Strikingly, the orm1 amiR-ORM2 plants showed increased expression of genes related to endoplasmic reticulum stress and defenses and also had enhanced resistance to oxidative stress and pathogen infection. Taken together, our findings indicate that ORMs interact with SPT to regulate sphingolipid homeostasis and play a pivotal role in environmental stress tolerance in plants. PMID:27923879

  3. Characterization of inositol phospho-sphingolipid-phospholipase C 1 (Isc1) in Cryptococcus neoformans reveals unique biochemical features.

    PubMed

    Henry, Jennifer; Guillotte, Aimee; Luberto, Chiara; Del Poeta, Maurizio

    2011-02-18

    In this work, we biochemically characterized inositol phosphosphingolipid-phospholipase C (Isc1) from the pathogenic fungus Cryptococcus neoformans. Unlike Isc1 from other fungi and parasites which hydrolyze both fungal complex sphingolipids (IPC-PLC) and mammalian sphingomyelin (SM-PLC), C. neoformans Isc1 only exerts IPC-PLC activity. Genetic mutations thought to regulate substrate recognition in other Isc1 proteins do not restore SM-PLC activity of the cryptococcal enzyme. C. neoformans Isc1 regulates the level of complex sphingolipids and certain species of phytoceramide, especially when fungal cells are exposed to acidic stress. Since growth in acidic environments is required for C. neoformans to cause disease, this study has important implications for understanding of C. neoformans pathogenicity.

  4. Factors to consider in using [U-C]palmitate for analysis of sphingolipid biosynthesis by tandem mass spectrometry[S

    PubMed Central

    Haynes, Christopher A.; Allegood, Jeremy C.; Wang, Elaine W.; Kelly, Samuel L.; Sullards, M. Cameron; Merrill, Alfred H.

    2011-01-01

    This study describes the use of a stable-isotope labeled precursor ([U-13C]palmitate) to analyze de novo sphingolipid biosynthesis by tandem mass spectrometry. It also describes factors to consider in interpreting the data, including the isotope's location (13C appears in three isotopomers and isotopologues: [M + 16] for the sphingoid base or N-acyl fatty acid, and [M + 32] for both); the isotopic enrichment of palmitoyl-CoA; and its elongation, desaturation, and incorporation into N-acyl-sphingolipids. For HEK293 cells incubated with 0.1 mM [U-13C]palmitic acid, ∼60% of the total palmitoyl-CoA was 13C-labeled by 3 h (which was near isotopic equilibrium); with this correction, the rates of de novo biosynthesis of C16:0-ceramide, C16:0-monohexosylceramide, and C16:0-sphingomyelins were 62 ± 3, 13 ± 2, and 60 ± 11 pmol/h per mg protein, respectively, which are consistent with an estimated rate of appearance of C16:0-ceramide using exponential growth modeling (119 ± 11 pmol/h per mg protein). Including estimates for the very long-chain fatty acyl-CoAs, the overall rate of sphingolipid biosynthesis can be estimated to be at least ∼1.6-fold higher. Thus, consideration of these factors gives a more accurate picture of de novo sphingolipid biosynthesis than has been possible to-date, while acknowledging that there are inherent limitations to such approximations. PMID:21586681

  5. Sphingolipid metabolism is a crucial determinant of cellular fate in nonstimulated proliferating Madin-Darby canine kidney (MDCK) cells.

    PubMed

    Nieto, Francisco Leocata; Pescio, Lucila G; Favale, Nicolás O; Adamo, Ana M; Sterin-Speziale, Norma B

    2008-09-12

    The present report was addressed to study the influence of sphingolipid metabolism in determining cellular fate. In nonstimulated proliferating Madin-Darby canine kidney (MDCK) cells, sphingolipid de novo synthesis is branched mainly to a production of sphingomyelin and ceramide, with a minor production of sphingosylphosphocholine, ceramide 1-phosphate, and sphingosine 1-phosphate. Experiments with (32)P as a radioactive precursor showed that sphingosine 1-phosphate is produced mainly by a de novo independent pathway. Enzymatic inhibition of the de novo pathway and ceramide synthesis affected cell number and viability only slightly, without changing sphingosine 1-phosphate production. By contrast, inhibition of sphingosine kinase-1 activity provoked a significant reduction in both cell number and viability in a dose-dependent manner. When sphingolipid metabolism was studied, an increase in de novo formed ceramide was found, which correlated with the concentration of enzyme inhibitor and the reduction in cell number and viability. Knockdown of sphingosine kinase-1 expression also induced an accumulation of de novo synthesized ceramide, provoking a slight reduction in cell number and viability similar to that induced by a low concentration of the sphingosine kinase inhibitor. Taken together, our results indicate that the level of de novo formed ceramide is controlled by the synthesis of sphingosine 1-phosphate, which appears to occur through a de novo synthesis-independent pathway, most probably the salvage pathway, that is responsible for the MDCK cell fate, suggesting that under proliferating conditions, a dynamic interplay exists between the de novo synthesis and the salvage pathway.

  6. Viral serine palmitoyltransferase induces metabolic switch in sphingolipid biosynthesis and is required for infection of a marine alga

    PubMed Central

    Ziv, Carmit; Malitsky, Sergey; Ben-Dor, Shifra; Wei, Yu; Zheng, Shuning; Aharoni, Asaph; Vardi, Assaf

    2016-01-01

    Marine viruses are the most abundant biological entities in the oceans shaping community structure and nutrient cycling. The interaction between the bloom-forming alga Emiliania huxleyi and its specific large dsDNA virus (EhV) is a major factor determining the fate of carbon in the ocean, thus serving as a key host-pathogen model system. The EhV genome encodes for a set of genes involved in the de novo sphingolipid biosynthesis, not reported in any viral genome to date. We combined detailed lipidomic and biochemical analyses to characterize the functional role of this virus-encoded pathway during lytic viral infection. We identified a major metabolic shift, mediated by differential substrate specificity of virus-encoded serine palmitoyltransferase, a key enzyme of sphingolipid biosynthesis. Consequently, unique viral glycosphingolipids, composed of unusual hydroxylated C17 sphingoid bases (t17:0) were highly enriched in the infected cells, and their synthesis was found to be essential for viral assembly. These findings uncover the biochemical bases of the virus-induced metabolic rewiring of the host sphingolipid biosynthesis during the chemical “arms race” in the ocean. PMID:26984500

  7. Alkaline Ceramidase 3 Deficiency Results in Purkinje Cell Degeneration and Cerebellar Ataxia Due to Dyshomeostasis of Sphingolipids in the Brain

    PubMed Central

    Preston, Chet; Wang, Louis; Yi, Jae Kyo; Lin, Chih-Li; Sun, Wei; Spyropoulos, Demetri D.; Rhee, Soyoung; Li, Mingsong; Zhou, Jie; Ge, Shaoyu; Zhang, Guofeng; Snider, Ashley J.; Hannun, Yusuf A.; Obeid, Lina M.; Mao, Cungui

    2015-01-01

    Dyshomeostasis of both ceramides and sphingosine-1-phosphate (S1P) in the brain has been implicated in aging-associated neurodegenerative disorders in humans. However, mechanisms that maintain the homeostasis of these bioactive sphingolipids in the brain remain unclear. Mouse alkaline ceramidase 3 (Acer3), which preferentially catalyzes the hydrolysis of C18:1-ceramide, a major unsaturated long-chain ceramide species in the brain, is upregulated with age in the mouse brain. Acer3 knockout causes an age-dependent accumulation of various ceramides and C18:1-monohexosylceramide and abolishes the age-related increase in the levels of sphingosine and S1P in the brain; thereby resulting in Purkinje cell degeneration in the cerebellum and deficits in motor coordination and balance. Our results indicate that Acer3 plays critically protective roles in controlling the homeostasis of various sphingolipids, including ceramides, sphingosine, S1P, and certain complex sphingolipids in the brain and protects Purkinje cells from premature degeneration. PMID:26474409

  8. Loss of Frataxin induces iron toxicity, sphingolipid synthesis, and Pdk1/Mef2 activation, leading to neurodegeneration

    PubMed Central

    Chen, Kuchuan; Lin, Guang; Haelterman, Nele A; Ho, Tammy Szu-Yu; Li, Tongchao; Li, Zhihong; Duraine, Lita; Graham, Brett H; Jaiswal, Manish; Yamamoto, Shinya; Rasband, Matthew N; Bellen, Hugo J

    2016-01-01

    Mutations in Frataxin (FXN) cause Friedreich’s ataxia (FRDA), a recessive neurodegenerative disorder. Previous studies have proposed that loss of FXN causes mitochondrial dysfunction, which triggers elevated reactive oxygen species (ROS) and leads to the demise of neurons. Here we describe a ROS independent mechanism that contributes to neurodegeneration in fly FXN mutants. We show that loss of frataxin homolog (fh) in Drosophila leads to iron toxicity, which in turn induces sphingolipid synthesis and ectopically activates 3-phosphoinositide dependent protein kinase-1 (Pdk1) and myocyte enhancer factor-2 (Mef2). Dampening iron toxicity, inhibiting sphingolipid synthesis by Myriocin, or reducing Pdk1 or Mef2 levels, all effectively suppress neurodegeneration in fh mutants. Moreover, increasing dihydrosphingosine activates Mef2 activity through PDK1 in mammalian neuronal cell line suggesting that the mechanisms are evolutionarily conserved. Our results indicate that an iron/sphingolipid/Pdk1/Mef2 pathway may play a role in FRDA. DOI: http://dx.doi.org/10.7554/eLife.16043.001 PMID:27343351

  9. Dihydroceramide Desaturase Knockdown Impacts Sphingolipids and Apoptosis after Photodamage in Human Head and Neck Squamous Carcinoma Cells

    PubMed Central

    BREEN, PAUL; JOSEPH, NICHOLAS; THOMPSON, KYLE; KRAVEKA, JACQUELINE M.; GUDZ, TATYANA I.; LI, LI; RAHMANIYAN, MEHRDAD; BIELAWSKI, JACEK; PIERCE, JASON S.; VAN BUREN, ERIC; BHATTI, GAURAV; SEPAROVIC, DUSKA

    2014-01-01

    Background Dihydroceramide desaturase 1 (DES) is the enzyme responsible for converting dihydroceramide into ceramide in the de novo sphingolipid biosynthesis pathway. Dihydroceramide can inhibit ceramide channel formation to interfere with apoptosis. We have shown that following ceramide synthase knockdown, photodynamic therapy (PDT), a cancer treatment modality, is associated with decreased levels of ceramides and dihydroceramides in cells that are resistant to apoptosis. Aim Here we investigated the effect of DES knockdown on the sphingolipid profile and apoptosis in human head and neck squamous carcinoma cells after PDT with the silicon phthalocyanine Pc 4. Materials and Methods Following siRNA transfection and PDT treatment, quantitative real-time polymerase chain reaction for quantification of DES mRNA, immunoblotting for protein expression, mass spectrometry for sphingolipid analysis, spectrofluorometry for caspase 3-like (DEVDase) activity, flow cytometry for apoptosis detection, and trypan blue assay for cell viability evaluation, were performed. Results Down-regulation of DES led to a substantial increase in levels of dihydroceramides without affecting ceramide levels. PDT-induced accumulation of individual dihydroceramides and global ceramides was increased by DES knockdown. Concomitantly, mitochondrial depolarization, DEVDase activation, late-apoptosis and cell death were attenuated by DES knockdown. Early apoptosis, however, was enhanced. Conclusion Our findings support the following: (i) dihydroceramide reduces pro-apoptotic effects of ceramide; (ii) cells adapt to DES knockdown to become more sensitive to ceramide and early-apoptosis; (iii) DES is a potential molecular target for regulating apoptotic resistance to PDT. PMID:23267130

  10. Plasma sphingolipids as potential indicators of hepatic necroinflammation in patients with chronic hepatitis C and normal alanine aminotransferase level.

    PubMed

    Li, Jun-Feng; Qu, Feng; Zheng, Su-Jun; Ren, Jin-Yu; Wu, Hui-Li; Liu, Mei; Liu, Hui; Ren, Feng; Chen, Yu; Zhang, Jin-Lan; Duan, Zhong-Ping

    2014-01-01

    Accurate estimation of hepatic necroinflammation caused by chronic hepatitis C (CHC) is crucial for prediction of prognosis and design of therapeutic strategy, which is particularly true for CHC patients with normal alanine aminotransferase (ALT) level. Recent studies have shown that sphingolipids have a close relationship with hepatitis C virus infection. The present study aimed to identify plasma sphingolipids related to hepatic necroinflammation. We included 120 treatment-naïve CHC patients and 64/120 had normal ALT levels (<40 U/L). CHC patients who underwent liver biopsies were subjected to Scheuer scoring analysis for scope of hepatic inflammation. Plasma sphingolipids were detected by high-performance liquid chromatography tandem mass spectrometry. Our results showed 44 plasma sphingolipids were detected altogether. Of all detected sphingolipids, hexosylceramide (HexCer) (d18∶1/22∶0) and HexCer (d18∶1/24∶0) showed a significant difference among G0/G1, G2, and G3/G4 (P<0.05). For identifying hepatic necroinflammation (G≥2), after adjusting other factors, the odds ratio (OR) of HexCer (d18∶1/22∶0) reached 1.01 (95% confidence interval [CI]: 1.00-1.02). Furthermore, the area under the curve (AUC) of HexCer (d18∶1/22∶0) was 0.7 (P = 0.01) and approached that of ALT (AUC = 0.78). However, in CHC patients with normal ALT, HexCer (d18∶1/22∶0) was an independent factor (OR: 1.02, 95% CI: 1.01-1.03) to identify the hepatic necroinflammation (G≥2). HexCer (d18∶1/22∶0) not only showed the largest AUC (0.78, P = 0.001), but also exhibited the highest specificity of all indicators. These results indicate that plasma HexCer (d18∶1/22∶0) is a potential indicator to distinguish hepatic necroinflammation in CHC patients. For CHC with normal ALT, the ability of HexCer (d18∶1/22∶0) to distinguish hepatic necroinflammation might be superior to conventional serum indicators.

  11. Plasma Sphingolipids as Potential Indicators of Hepatic Necroinflammation in Patients with Chronic Hepatitis C and Normal Alanine Aminotransferase Level

    PubMed Central

    Ren, Jin-Yu; Wu, Hui-Li; Liu, Mei; Liu, Hui; Ren, Feng; Chen, Yu; Zhang, Jin-Lan; Duan, Zhong-Ping

    2014-01-01

    Accurate estimation of hepatic necroinflammation caused by chronic hepatitis C (CHC) is crucial for prediction of prognosis and design of therapeutic strategy, which is particularly true for CHC patients with normal alanine aminotransferase (ALT) level. Recent studies have shown that sphingolipids have a close relationship with hepatitis C virus infection. The present study aimed to identify plasma sphingolipids related to hepatic necroinflammation. We included 120 treatment-naïve CHC patients and 64/120 had normal ALT levels (<40 U/L). CHC patients who underwent liver biopsies were subjected to Scheuer scoring analysis for scope of hepatic inflammation. Plasma sphingolipids were detected by high-performance liquid chromatography tandem mass spectrometry. Our results showed 44 plasma sphingolipids were detected altogether. Of all detected sphingolipids, hexosylceramide (HexCer) (d18∶1/22∶0) and HexCer (d18∶1/24∶0) showed a significant difference among G0/G1, G2, and G3/G4 (P<0.05). For identifying hepatic necroinflammation (G≥2), after adjusting other factors, the odds ratio (OR) of HexCer (d18∶1/22∶0) reached 1.01 (95% confidence interval [CI]: 1.00–1.02). Furthermore, the area under the curve (AUC) of HexCer (d18∶1/22∶0) was 0.7 (P = 0.01) and approached that of ALT (AUC = 0.78). However, in CHC patients with normal ALT, HexCer (d18∶1/22∶0) was an independent factor (OR: 1.02, 95% CI: 1.01–1.03) to identify the hepatic necroinflammation (G≥2). HexCer (d18∶1/22∶0) not only showed the largest AUC (0.78, P = 0.001), but also exhibited the highest specificity of all indicators. These results indicate that plasma HexCer (d18∶1/22∶0) is a potential indicator to distinguish hepatic necroinflammation in CHC patients. For CHC with normal ALT, the ability of HexCer (d18∶1/22∶0) to distinguish hepatic necroinflammation might be superior to conventional serum indicators. PMID:24736528

  12. Bladder cancer cell growth and motility implicate cannabinoid 2 receptor-mediated modifications of sphingolipids metabolism

    PubMed Central

    Bettiga, Arianna; Aureli, Massimo; Colciago, Giorgia; Murdica, Valentina; Moschini, Marco; Lucianò, Roberta; Canals, Daniel; Hannun, Yusuf; Hedlund, Petter; Lavorgna, Giovanni; Colombo, Renzo; Bassi, Rosaria; Samarani, Maura; Montorsi, Francesco; Salonia, Andrea; Benigni, Fabio

    2017-01-01

    The inhibitory effects demonstrated by activation of cannabinoid receptors (CB) on cancer proliferation and migration may also play critical roles in controlling bladder cancer (BC). CB expression on human normal and BC specimens was tested by immunohistochemistry. Human BC cells RT4 and RT112 were challenged with CB agonists and assessed for proliferation, apoptosis, and motility. Cellular sphingolipids (SL) constitution and metabolism were evaluated after metabolic labelling. CB1-2 were detected in BC specimens, but only CB2 was more expressed in the tumour. Both cell lines expressed similar CB2. Exposure to CB2 agonists inhibited BC growth, down-modulated Akt, induced caspase 3-activation and modified SL metabolism. Baseline SL analysis in cell lines showed differences linked to unique migratory behaviours and cytoskeletal re-arrangements. CB2 activation changed the SL composition of more aggressive RT112 cells by reducing (p < 0.01) Gb3 ganglioside (−50 ± 3%) and sphingosine 1-phosphate (S1P, −40 ± 4%), which ended up to reduction in cell motility (−46 ± 5%) with inhibition of p-SRC. CB2-selective antagonists, gene silencing and an inhibitor of SL biosynthesis partially prevented CB2 agonist-induced effects on cell viability and motility. CB2 activation led to ceramide-mediated BC cell apoptosis independently of SL constitutive composition, which instead was modulated by CB2 agonists to reduce cell motility. PMID:28191815

  13. Structure and function of sphingosine-1-phosphate lyase, a key enzyme of sphingolipid metabolism.

    PubMed

    Bourquin, Florence; Riezman, Howard; Capitani, Guido; Grütter, Markus G

    2010-08-11

    Sphingosine-1-phosphate lyase (SPL), a key enzyme of sphingolipid metabolism, catalyzes the irreversible degradation of sphingoid base phosphates. Its main substrate sphingosine-1-phosphate (S1P) acts both extracellularly, by binding G protein-coupled receptors of the lysophospholipid receptor family, and inside the cell, as a second messenger. There, S1P takes part in regulating various cellular processes and its levels are tightly regulated. SPL is a pivotal enzyme regulating S1P intracellular concentrations and a promising drug target for the design of immunosuppressants. We structurally and functionally characterized yeast SPL (Dpl1p) and its first prokaryotic homolog, from Symbiobacterium thermophilum. The Dpl1p structure served as a basis for a very reliable model of Homo sapiens SPL. The above results, together with in vitro and in vivo studies of SPL mutants, reveal which residues are involved in activity and substrate binding and pave the way to studies aimed at controlling the activity of this pivotal enzyme.

  14. Efficient replacement of plasma membrane outer leaflet phospholipids and sphingolipids in cells with exogenous lipids.

    PubMed

    Li, Guangtao; Kim, JiHyun; Huang, Zhen; St Clair, Johnna R; Brown, Deborah A; London, Erwin

    2016-12-06

    Our understanding of membranes and membrane lipid function has lagged far behind that of nucleic acids and proteins, largely because it is difficult to manipulate cellular membrane lipid composition. To help solve this problem, we show that methyl-α-cyclodextrin (MαCD)-catalyzed lipid exchange can be used to maximally replace the sphingolipids and phospholipids in the outer leaflet of the plasma membrane of living mammalian cells with exogenous lipids, including unnatural lipids. In addition, lipid exchange experiments revealed that 70-80% of cell sphingomyelin resided in the plasma membrane outer leaflet; the asymmetry of metabolically active cells was similar to that previously defined for erythrocytes, as judged by outer leaflet lipid composition; and plasma membrane outer leaflet phosphatidylcholine had a significantly lower level of unsaturation than phosphatidylcholine in the remainder of the cell. The data also provided a rough estimate for the total cellular lipids residing in the plasma membrane (about half). In addition to such lipidomics applications, the exchange method should have wide potential for investigations of lipid function and modification of cellular behavior by modification of lipids.

  15. Male meiotic cytokinesis requires ceramide synthase 3-dependent sphingolipids with unique membrane anchors.

    PubMed

    Rabionet, Mariona; Bayerle, Aline; Jennemann, Richard; Heid, Hans; Fuchser, Jens; Marsching, Christian; Porubsky, Stefan; Bolenz, Christian; Guillou, Florian; Gröne, Hermann-Josef; Gorgas, Karin; Sandhoff, Roger

    2015-09-01

    Somatic cell cytokinesis was shown to involve the insertion of sphingolipids (SLs) to midbodies prior to abscission. Spermatogenic midbodies transform into stable intercellular bridges (ICBs) connecting clonal daughter cells in a syncytium. This process requires specialized SL structures. (1) Using high resolution-mass spectrometric imaging, we show in situ a biphasic pattern of SL synthesis with testis-specific anchors. This pattern correlates with and depends on ceramide synthase 3 (CerS3) localization in both, pachytene spermatocytes until completion of meiosis and elongating spermatids. (2) Blocking the pathways to germ cell-specific ceramides (CerS3-KO) and further to glycosphingolipids (glucosylceramide synthase-KO) in mice highlights the need for special SLs for spermatid ICB stability. In contrast to somatic mitosis these SLs require ultra-long polyunsaturated anchors with unique physico-chemical properties, which can only be provided by CerS3. Loss of these anchors causes enhanced apoptosis during meiosis, formation of multinuclear giant cells and spermatogenic arrest. Hence, testis-specific SLs, which we also link to CerS3 in human testis, are quintessential for male fertility. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  16. Identification of a New Class of Antifungals Targeting the Synthesis of Fungal Sphingolipids

    PubMed Central

    Mor, Visesato; Rella, Antonella; Farnoud, Amir M.; Singh, Ashutosh; Munshi, Mansa; Bryan, Arielle; Naseem, Shamoon; Konopka, James B.; Ojima, Iwao; Bullesbach, Erika; Ashbaugh, Alan; Linke, Michael J.; Cushion, Melanie; Collins, Margaret; Ananthula, Hari Krishna; Sallans, Larry; Desai, Pankaj B.; Wiederhold, Nathan P.; Fothergill, Annette W.; Kirkpatrick, William R.; Patterson, Thomas; Wong, Lai Hong; Sinha, Sunita; Giaever, Guri; Nislow, Corey; Flaherty, Patrick; Pan, Xuewen; Cesar, Gabriele Vargas; de Melo Tavares, Patricia; Frases, Susana; Miranda, Kildare; Rodrigues, Marcio L.; Luberto, Chiara; Nimrichter, Leonardo

    2015-01-01

    ABSTRACT Recent estimates suggest that >300 million people are afflicted by serious fungal infections worldwide. Current antifungal drugs are static and toxic and/or have a narrow spectrum of activity. Thus, there is an urgent need for the development of new antifungal drugs. The fungal sphingolipid glucosylceramide (GlcCer) is critical in promoting virulence of a variety of human-pathogenic fungi. In this study, we screened a synthetic drug library for compounds that target the synthesis of fungal, but not mammalian, GlcCer and found two compounds [N′-(3-bromo-4-hydroxybenzylidene)-2-methylbenzohydrazide (BHBM) and its derivative, 3-bromo-N′-(3-bromo-4-hydroxybenzylidene) benzohydrazide (D0)] that were highly effective in vitro and in vivo against several pathogenic fungi. BHBM and D0 were well tolerated in animals and are highly synergistic or additive to current antifungals. BHBM and D0 significantly affected fungal cell morphology and resulted in the accumulation of intracellular vesicles. Deep-sequencing analysis of drug-resistant mutants revealed that four protein products, encoded by genes APL5, COS111, MKK1, and STE2, which are involved in vesicular transport and cell cycle progression, are targeted by BHBM. PMID:26106079

  17. Amyotrophic lateral sclerosis and denervation alter sphingolipids and up-regulate glucosylceramide synthase

    PubMed Central

    Henriques, Alexandre; Croixmarie, Vincent; Priestman, David A.; Rosenbohm, Angela; Dirrig-Grosch, Sylvie; D'Ambra, Eleonora; Huebecker, Mylene; Hussain, Ghulam; Boursier-Neyret, Claire; Echaniz-Laguna, Andoni; Ludolph, Albert C.; Platt, Frances M.; Walther, Bernard; Spedding, Michael; Loeffler, Jean-Philippe; Gonzalez De Aguilar, Jose-Luis

    2015-01-01

    Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset disease characterized by upper and lower motor neuron degeneration, muscle wasting and paralysis. Growing evidence suggests a link between changes in lipid metabolism and ALS. Here, we used UPLC/TOF-MS to survey the lipidome in SOD1(G86R) mice, a model of ALS. Significant changes in lipid expression were evident in spinal cord and skeletal muscle before overt neuropathology. In silico analysis also revealed appreciable changes in sphingolipids including ceramides and glucosylceramides (GlcCer). HPLC analysis showed increased amounts of GlcCer and downstream glycosphingolipids (GSLs) in SOD1(G86R) muscle compared with wild-type littermates. Glucosylceramide synthase (GCS), the enzyme responsible for GlcCer biosynthesis, was up-regulated in muscle of SOD1(G86R) mice and ALS patients, and in muscle of wild-type mice after surgically induced denervation. Conversely, inhibition of GCS in wild-type mice, following transient peripheral nerve injury, reversed the overexpression of genes in muscle involved in oxidative metabolism and delayed motor recovery. GCS inhibition in SOD1(G86R) mice also affected the expression of metabolic genes and induced a loss of muscle strength and morphological deterioration of the motor endplates. These findings suggest that GSLs may play a critical role in ALS muscle pathology and could lead to the identification of new therapeutic targets. PMID:26483191

  18. Changes in membrane sphingolipid composition modulate dynamics and adhesion of integrin nanoclusters

    PubMed Central

    Eich, Christina; Manzo, Carlo; Keijzer, Sandra de; Bakker, Gert-Jan; Reinieren-Beeren, Inge; García-Parajo, Maria F.; Cambi, Alessandra

    2016-01-01

    Sphingolipids are essential constituents of the plasma membrane (PM) and play an important role in signal transduction by modulating clustering and dynamics of membrane receptors. Changes in lipid composition are therefore likely to influence receptor organisation and function, but how this precisely occurs is difficult to address given the intricacy of the PM lipid-network. Here, we combined biochemical assays and single molecule dynamic approaches to demonstrate that the local lipid environment regulates adhesion of integrin receptors by impacting on their lateral mobility. Induction of sphingomyelinase (SMase) activity reduced sphingomyelin (SM) levels by conversion to ceramide (Cer), resulting in impaired integrin adhesion and reduced integrin mobility. Dual-colour imaging of cortical actin in combination with single molecule tracking of integrins showed that this reduced mobility results from increased coupling to the actin cytoskeleton brought about by Cer formation. As such, our data emphasizes a critical role for the PM local lipid composition in regulating the lateral mobility of integrins and their ability to dynamically increase receptor density for efficient ligand binding in the process of cell adhesion. PMID:26869100

  19. A novel sphingolipid-TORC1 pathway critically promotes postembryonic development in Caenorhabditis elegans

    PubMed Central

    Zhu, Huanhu; Shen, Huali; Sewell, Aileen K; Kniazeva, Marina; Han, Min

    2013-01-01

    Regulation of animal development in response to nutritional cues is an intensely studied problem related to disease and aging. While extensive studies indicated roles of the Target of Rapamycin (TOR) in sensing certain nutrients for controlling growth and metabolism, the roles of fatty acids and lipids in TOR-involved nutrient/food responses are obscure. Caenorhabditis elegans halts postembryonic growth and development shortly after hatching in response to monomethyl branched-chain fatty acid (mmBCFA) deficiency. Here, we report that an mmBCFA-derived sphingolipid, d17iso-glucosylceramide, is a critical metabolite in regulating growth and development. Further analysis indicated that this lipid function is mediated by TORC1 and antagonized by the NPRL-2/3 complex in the intestine. Strikingly, the essential lipid function is bypassed by activating TORC1 or inhibiting NPRL-2/3. Our findings uncover a novel lipid-TORC1 signaling pathway that coordinates nutrient and metabolic status with growth and development, advancing our understanding of the physiological roles of mmBCFAs, ceramides, and TOR. DOI: http://dx.doi.org/10.7554/eLife.00429.001 PMID:23705068

  20. The role of sphingolipid signalling in diabetes-associated pathologies (Review)

    PubMed Central

    Ng, Mei Li; Wadham, Carol; Sukocheva, Olga A.

    2017-01-01

    Sphingosine kinase (SphK) is an important signalling enzyme that catalyses the phosphorylation of sphingosine (Sph) to form sphingosine-1-phosphate (S1P). The multifunctional lipid, S1P binds to a family of five G protein-coupled receptors (GPCRs). As an intracellular second messenger, S1P activates key signalling cascades responsible for the maintenance of sphingolipid metabolism, and has been implicated in the progression of cancer, and the development of other inflammatory and metabolic diseases. SphK and S1P are critical molecules involved in the regulation of various cellular metabolic processes, such as cell proliferation, survival, apoptosis, adhesion and migration. There is strong evidence supporting the critical roles of SphK and S1P in the progression of diabetes mellitus, including insulin sensitivity and insulin secretion, pancreatic β-cell apoptosis, and the development of diabetic inflammatory state. In this review, we summarise the current state of knowledge for SphK/S1P signalling effects, associated with the development of insulin resistance, pancreatic β-cell death and the vascular complications of diabetes mellitus. PMID:28075451

  1. Efficient replacement of plasma membrane outer leaflet phospholipids and sphingolipids in cells with exogenous lipids

    PubMed Central

    Kim, JiHyun; Huang, Zhen; St. Clair, Johnna R.; Brown, Deborah A.; London, Erwin

    2016-01-01

    Our understanding of membranes and membrane lipid function has lagged far behind that of nucleic acids and proteins, largely because it is difficult to manipulate cellular membrane lipid composition. To help solve this problem, we show that methyl-α-cyclodextrin (MαCD)-catalyzed lipid exchange can be used to maximally replace the sphingolipids and phospholipids in the outer leaflet of the plasma membrane of living mammalian cells with exogenous lipids, including unnatural lipids. In addition, lipid exchange experiments revealed that 70–80% of cell sphingomyelin resided in the plasma membrane outer leaflet; the asymmetry of metabolically active cells was similar to that previously defined for erythrocytes, as judged by outer leaflet lipid composition; and plasma membrane outer leaflet phosphatidylcholine had a significantly lower level of unsaturation than phosphatidylcholine in the remainder of the cell. The data also provided a rough estimate for the total cellular lipids residing in the plasma membrane (about half). In addition to such lipidomics applications, the exchange method should have wide potential for investigations of lipid function and modification of cellular behavior by modification of lipids. PMID:27872310

  2. Revisiting Plant Plasma Membrane Lipids in Tobacco: A Focus on Sphingolipids1

    PubMed Central

    Cacas, Jean-Luc; Grosjean, Kevin; Gerbeau-Pissot, Patricia; Lherminier, Jeannine; Rombouts, Yoann; Maes, Emmanuel; Gronnier, Julien; Furt, Fabienne; Fouillen, Laetitia; Bayer, Emmanuelle; Cluzet, Stéphanie; Schmitter, Jean-Marie; Deleu, Magali; Lins, Laurence; Simon-Plas, Françoise; Mongrand, Sébastien

    2016-01-01

    The lipid composition of plasma membrane (PM) and the corresponding detergent-insoluble membrane (DIM) fraction were analyzed with a specific focus on highly polar sphingolipids, so-called glycosyl inositol phosphorylceramides (GIPCs). Using tobacco (Nicotiana tabacum) ‘Bright Yellow 2’ cell suspension and leaves, evidence is provided that GIPCs represent up to 40 mol % of the PM lipids. Comparative analysis of DIMs with the PM showed an enrichment of 2-hydroxylated very-long-chain fatty acid-containing GIPCs and polyglycosylated GIPCs in the DIMs. Purified antibodies raised against these GIPCs were further used for immunogold-electron microscopy strategy, revealing the distribution of polyglycosylated GIPCs in domains of 35 ± 7 nm in the plane of the PM. Biophysical studies also showed strong interactions between GIPCs and sterols and suggested a role for very-long-chain fatty acids in the interdigitation between the two PM-composing monolayers. The ins and outs of lipid asymmetry, raft formation, and interdigitation in plant membrane biology are finally discussed. PMID:26518342

  3. Inactivation of Ceramide Synthase 6 in Mice Results in an Altered Sphingolipid Metabolism and Behavioral Abnormalities*

    PubMed Central

    Ebel, Philipp; vom Dorp, Katharina; Petrasch-Parwez, Elisabeth; Zlomuzica, Armin; Kinugawa, Kiyoka; Mariani, Jean; Minich, David; Ginkel, Christina; Welcker, Jochen; Degen, Joachim; Eckhardt, Matthias; Dere, Ekrem; Dörmann, Peter; Willecke, Klaus

    2013-01-01

    The N-acyl chain length of ceramides is determined by the specificity of different ceramide synthases (CerS). The CerS family in mammals consists of six members with different substrate specificities and expression patterns. We have generated and characterized a mouse line harboring an enzymatically inactive ceramide synthase 6 (CerS6KO) gene and lacz reporter cDNA coding for β-galactosidase directed by the CerS6 promoter. These mice display a decrease in C16:0 containing sphingolipids. Relative to wild type tissues the amount of C16:0 containing sphingomyelin in kidney is ∼35%, whereas we find a reduction of C16:0 ceramide content in the small intestine to about 25%. The CerS6KO mice show behavioral abnormalities including a clasping abnormality of their hind limbs and a habituation deficit. LacZ reporter expression in the brain reveals CerS6 expression in hippocampus, cortex, and the Purkinje cell layer of the cerebellum. Using newly developed antibodies that specifically recognize the CerS6 protein we show that the endogenous CerS6 protein is N-glycosylated and expressed in several tissues of mice, mainly kidney, small and large intestine, and brain. PMID:23760501

  4. A minimalist approach to MALDI imaging of glycerophospholipids and sphingolipids in rat brain sections

    NASA Astrophysics Data System (ADS)

    Wang, Hay-Yan J.; Post, Shelley N. Jackson Jeremy; Woods, Amina S.

    2008-12-01

    Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a powerful tool that has allowed researchers to directly probe tissue molecular structure and drug content with minimal manipulations, while maintaining anatomical integrity. In the present work glycerophospholipids and sphingolipids images were acquired from 16-[mu]m thick coronal rat brain sections using MALDI-MS. Images of phosphatidylinositol 38:4 (PI 38:4), sulfatide 24:1 (ST 24:1), and hydroxyl sulfatide 24:1 (ST 24:1 (OH)) were acquired in negative ion mode, while the images of phosphatidylcholine 34:1 (PC 34:1), potassiated phosphatidylcholines 32:0 (PC 32:0 + K+) and 36:1 (PC 36:1 + K+) were acquired in positive ion mode. The images of PI 38:4 and PC 36:1 + K+ show the preferential distribution of these two lipids in gray matter; and the images of two sulfatides and PC 32:0 + K+ show their preferential distribution in white matter. In addition, the gray cortical band and its adjacent anatomical structures were also identified by contrasting their lipid makeup. The resulting images were compared to lipid images acquired by secondary ion mass spectrometry (SIMS). The suitability of TLC sprayers, Collison Nebulizer, and artistic airbrush were also evaluated as means for matrix deposition.

  5. Counter-regulation of opioid analgesia by glial-derived bioactive sphingolipids

    PubMed Central

    Muscoli, Carolina; Doyle, Tim; Dagostino, Concetta; Bryant, Leesa; Chen, Zhoumou; Watkins, Linda R.; Ryerse, Jan; Bieberich, Erhard; Neumman, William; Salvemini, Daniela

    2010-01-01

    The clinical efficacy of opiates for pain control is severely limited by analgesic tolerance and hyperalgesia. Herein we show that chronic morphine upregulates both the sphingolipid ceramide in spinal astrocytes and microglia, but not neurons, and spinal sphingosine-1-phosphate (S1P), the end-product of ceramide metabolism. Co-administering morphine with intrathecal administration of pharmacological inhibitors of ceramide and S1P blocked formation of spinal S1P and development of hyperalgesia and tolerance in rats. Our results show that spinally formed S1P signals at least in part by 1) modulating glial function, since inhibiting S1P formation blocked increased formation of glial-related pro-inflammatory cytokines, in particular TNF-α, IL-1β α, and IL6, which are known modulators of neuronal excitability, and 2) peroxynitrite-mediated post-translational nitration and inactivation of glial-related enzymes (glutamine synthetase and the glutamate transporter, GLT-1) known to play critical roles in glutamate neurotransmission. Inhibitors of the ceramide metabolic pathway may have therapeutic potential as adjuncts to opiates in relieving suffering from chronic pain. PMID:21084596

  6. Sphingolipids, Transcription Factors, and Conserved Toolkit Genes: Developmental Plasticity in the Ant Cardiocondyla obscurior

    PubMed Central

    Schrader, Lukas; Simola, Daniel F.; Heinze, Jürgen; Oettler, Jan

    2015-01-01

    Developmental plasticity allows for the remarkable morphological specialization of individuals into castes in eusocial species of Hymenoptera. Developmental trajectories that lead to alternative caste fates are typically determined by specific environmental stimuli that induce larvae to express and maintain distinct gene expression patterns. Although most eusocial species express two castes, queens and workers, the ant Cardiocondyla obscurior expresses diphenic females and males; this provides a unique system with four discrete phenotypes to study the genomic basis of developmental plasticity in ants. We sequenced and analyzed the transcriptomes of 28 individual C. obscurior larvae of known developmental trajectory, providing the first in-depth analysis of gene expression in eusocial insect larvae. Clustering and transcription factor binding site analyses revealed that different transcription factors and functionally distinct sets of genes are recruited during larval development to induce the four alternative trajectories. In particular, we found complex patterns of gene regulation pertaining to sphingolipid metabolism, a conserved molecular pathway involved in development, obesity, and aging. PMID:25725431

  7. Inactivation of ceramide synthase 6 in mice results in an altered sphingolipid metabolism and behavioral abnormalities.

    PubMed

    Ebel, Philipp; Vom Dorp, Katharina; Petrasch-Parwez, Elisabeth; Zlomuzica, Armin; Kinugawa, Kiyoka; Mariani, Jean; Minich, David; Ginkel, Christina; Welcker, Jochen; Degen, Joachim; Eckhardt, Matthias; Dere, Ekrem; Dörmann, Peter; Willecke, Klaus

    2013-07-19

    The N-acyl chain length of ceramides is determined by the specificity of different ceramide synthases (CerS). The CerS family in mammals consists of six members with different substrate specificities and expression patterns. We have generated and characterized a mouse line harboring an enzymatically inactive ceramide synthase 6 (CerS6KO) gene and lacz reporter cDNA coding for β-galactosidase directed by the CerS6 promoter. These mice display a decrease in C16:0 containing sphingolipids. Relative to wild type tissues the amount of C16:0 containing sphingomyelin in kidney is ∼35%, whereas we find a reduction of C16:0 ceramide content in the small intestine to about 25%. The CerS6KO mice show behavioral abnormalities including a clasping abnormality of their hind limbs and a habituation deficit. LacZ reporter expression in the brain reveals CerS6 expression in hippocampus, cortex, and the Purkinje cell layer of the cerebellum. Using newly developed antibodies that specifically recognize the CerS6 protein we show that the endogenous CerS6 protein is N-glycosylated and expressed in several tissues of mice, mainly kidney, small and large intestine, and brain.

  8. Digestion of Ceramide 2-Aminoethylphosphonate, a Sphingolipid from the Jumbo Flying Squid Dosidicus gigas, in Mice.

    PubMed

    Tomonaga, Nami; Manabe, Yuki; Sugawara, Tatsuya

    2017-04-01

    Ceramide 2-aminoethylphosphonate (CAEP), a sphingophosphonolipid containing a carbon-phosphorus bond, is frequently found in marine organisms and has a unique triene type of sphingoid base in its structure. CAEP has not been evaluated as a food ingredient, although it is generally contained in Mollusca organisms such as squids and shellfish, which are consumed worldwide. In this study, we aimed to elucidate the effects of CAEP as a food component by evaluating the digestion of CAEP extracted from the skin of the jumbo flying squid Dosidicus gigas. Our results revealed that dietary CAEP was digested to free sphingoid bases via ceramides by the mouse small intestinal mucosa. At pH 7.2, CAEP was hydrolyzed more rapidly than the major mammalian sphingolipid sphingomyelin; however, the hydrolysis of CAEP was similar to that of sphingomyelin at pH 9.0. Thus, the digestion of CAEP may be catalyzed by alkaline spingomyelinase and other enzymes. Our findings provide important insights into the digestion of the dietary sphingophosphonolipid CAEP in marine foods.

  9. From Molecules to the Clinic: Linking Schizophrenia and Metabolic Syndrome through Sphingolipids Metabolism

    PubMed Central

    Castillo, Rolando I.; Rojo, Leonel E.; Henriquez-Henriquez, Marcela; Silva, Hernán; Maturana, Alejandro; Villar, María J.; Fuentes, Manuel; Gaspar, Pablo A.

    2016-01-01

    Metabolic syndrome (MS) is a prevalent and severe comorbidity observed in schizophrenia (SZ). The exact nature of this association is controversial and very often accredited to the effects of psychotropic medications and disease-induced life-style modifications, such as inactive lifestyle, poor dietary choices, and smoking. However, drug therapy and disease-induced lifestyle factors are likely not the only factors contributing to the observed converging nature of these conditions, since an increased prevalence of MS is also observed in first episode and drug-naïve psychosis populations. MS and SZ share common intrinsic susceptibility factors and etiopathogenic mechanisms, which may change the way we approach clinical management of SZ patients. Among the most relevant common pathogenic pathways of SZ and MS are alterations in the sphingolipids (SLs) metabolism and SLs homeostasis. SLs have important structural functions as they participate in the formation of membrane “lipid rafts.” SLs also play physiological roles in cell differentiation, proliferation, and inflammatory processes, which might be part of MS/SZ common pathophysiological processes. In this article we review a plausible mechanism to explain the link between MS and SZ through a disruption in SL homeostasis. Additionally, we provide insights on how this hypothesis can lead to the developing of new diagnostic/therapeutic technologies for SZ patients. PMID:27877101

  10. Fast screening of highly glycosylated plant sphingolipids by tandem mass spectrometry.

    PubMed

    Buré, Corinne; Cacas, Jean-Luc; Wang, Fen; Gaudin, Karen; Domergue, Frédéric; Mongrand, Sébastien; Schmitter, Jean-Marie

    2011-10-30

    The structural characterization of Glycosyl-Inositol-Phospho-Ceramides (GIPCs), which are the main sphingolipids of plant tissues, is a critical step towards the understanding of their physiological function. After optimization of their extraction, numerous plant GIPCs have been characterized by mass spectrometry. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) full scan analysis of negative ions provides a quick overview of GIPC distribution. Clear differences were observed for the two plant models studied: six GIPC series bearing from two to seven saccharide units were detected in tobacco BY-2 cell extracts, whereas GIPCs extracted from A. thaliana cell cultures and leaves were less diverse, with a dominance of species containing only two saccharide units. The number of GIPC species was around 50 in A. thaliana and 120 in tobacco BY-2 cells. MALDI-MS/MS spectra gave access to detailed structural information relative to the ceramide moiety, the polar head, as well as the number and types of saccharide units. Once released from GIPCs, fatty acid chains and long-chain bases were analyzed by GC/MS to verify that all GIPC series were taken into account by the MALDI-MS/MS approach. ESI-MS/MS provided complementary information for the identification of isobaric species and fatty acid chains. Such a methodology, mostly relying on MALDI-MS/MS, should open new avenues to determine structure-function relationships between glycosphingolipids and membrane organization.

  11. From Molecules to the Clinic: Linking Schizophrenia and Metabolic Syndrome through Sphingolipids Metabolism.

    PubMed

    Castillo, Rolando I; Rojo, Leonel E; Henriquez-Henriquez, Marcela; Silva, Hernán; Maturana, Alejandro; Villar, María J; Fuentes, Manuel; Gaspar, Pablo A

    2016-01-01

    Metabolic syndrome (MS) is a prevalent and severe comorbidity observed in schizophrenia (SZ). The exact nature of this association is controversial and very often accredited to the effects of psychotropic medications and disease-induced life-style modifications, such as inactive lifestyle, poor dietary choices, and smoking. However, drug therapy and disease-induced lifestyle factors are likely not the only factors contributing to the observed converging nature of these conditions, since an increased prevalence of MS is also observed in first episode and drug-naïve psychosis populations. MS and SZ share common intrinsic susceptibility factors and etiopathogenic mechanisms, which may change the way we approach clinical management of SZ patients. Among the most relevant common pathogenic pathways of SZ and MS are alterations in the sphingolipids (SLs) metabolism and SLs homeostasis. SLs have important structural functions as they participate in the formation of membrane "lipid rafts." SLs also play physiological roles in cell differentiation, proliferation, and inflammatory processes, which might be part of MS/SZ common pathophysiological processes. In this article we review a plausible mechanism to explain the link between MS and SZ through a disruption in SL homeostasis. Additionally, we provide insights on how this hypothesis can lead to the developing of new diagnostic/therapeutic technologies for SZ patients.

  12. De Novo sphingolipid synthesis is essential for Salmonella-induced autophagy and human beta-defensin 2 expression in intestinal epithelial cells.

    PubMed

    Huang, Fu-Chen

    2016-01-01

    Sphingolipids are important for innate immune response to eliminate infected pathogens and involved in autophagy. On the other hand, nucleotide-binding oligomerization domain-containing protein 2 (NOD2) served as an intracellular pattern recognition receptor to enhance host defense by inducing autophagy and the production of antimicrobial peptides, such as human beta-defensin-2 (hBD-2). However, the role of sphingolipids in Salmonella-induced autophagy and hBD-2 response in intestinal epithelial cells has not been previously elucidated. Salmonella typhimurium wild-type strain SL1344 was used to infect SW480, an intestinal epithelial cell. hBD-2 and interleukin-8 (IL-8) mRNA expressions were assessed in SW480 cells using RT-PCR, and intracellular signaling pathways and autophagy protein expression were analyzed by Western blot in SW480 cells in the presence or absence of inhibitors or transfected with siRNA. We demonstrated that inhibition of de novo sphingolipid synthesis repressed the membrane recruitment of NOD2 and autophagy-related protein 16-like 1 (Atg16L1), suppressed Salmonella-induced autophagic protein LC3-II expression, and reduced NOD2-mediated hBD-2 response in Salmonella-infected SW480 cells. Contrasting to the utilization of membrane cholesterol on maintenance of Salmonella-containing vacuoles and anti-inflammation by Salmonella, sphingolipids act on epithelial defense against the invasive pathogen. Our results offer mechanistic insights on the role of de novo sphingolipid synthesis in the innate immunity of intestinal epithelial cells to Salmonella infection. The pharmaceuticals enhancing or diet enriched with sphingolipids may induce the dual anti-bacterial mechanisms. The role of de novo sphingolipid synthesis on inflammatory bowel disease is deserved to be further investigated.

  13. Dysregulations in circulating sphingolipids associate with disease activity indices in female patients with systemic lupus erythematosus: a cross-sectional study.

    PubMed

    Checa, A; Idborg, H; Zandian, A; Sar, D Garcia; Surowiec, I; Trygg, J; Svenungsson, E; Jakobsson, P-J; Nilsson, P; Gunnarsson, I; Wheelock, C E

    2017-01-01

    Objective The objective of this study was to investigate the association of clinical and renal disease activity with circulating sphingolipids in patients with systemic lupus erythematosus. Methods We used liquid chromatography tandem mass spectrometry to measure the levels of 27 sphingolipids in plasma from 107 female systemic lupus erythematosus patients and 23 controls selected using a design of experiment approach. We investigated the associations between sphingolipids and two disease activity indices, the Systemic Lupus Activity Measurement and the Systemic Lupus Erythematosus Disease Activity Index. Damage was scored according to the Systemic Lupus International Collaborating Clinics damage index. Renal activity was evaluated with the British Island Lupus Activity Group index. The effects of immunosuppressive treatment on sphingolipid levels were evaluated before and after treatment in 22 female systemic lupus erythematosus patients with active disease. Results Circulating sphingolipids from the ceramide and hexosylceramide families were increased, and sphingoid bases were decreased, in systemic lupus erythematosus patients compared to controls. The ratio of C16:0-ceramide to sphingosine-1-phosphate was the best discriminator between patients and controls, with an area under the receiver-operating curve of 0.77. The C16:0-ceramide to sphingosine-1-phosphate ratio was associated with ongoing disease activity according to the Systemic Lupus Activity Measurement and the Systemic Lupus Erythematosus Disease Activity Index, but not with accumulated damage according to the Systemic Lupus International Collaborating Clinics Damage Index. Levels of C16:0- and C24:1-hexosylceramides were able to discriminate patients with current versus inactive/no renal involvement. All dysregulated sphingolipids were normalized after immunosuppressive treatment. Conclusion We provide evidence that sphingolipids are dysregulated in systemic lupus erythematosus and associated with

  14. Delineating Amyloid Plaque Associated Neuronal Sphingolipids in Transgenic Alzheimer’s Disease Mice (tgArcSwe) Using MALDI Imaging Mass Spectrometry

    PubMed Central

    2016-01-01

    The major pathological hallmarks of Alzheimer’s disease (AD) are the progressive aggregation and accumulation of beta-amyloid (Aβ) and hyperphosphorylated tau protein into neurotoxic deposits. Aβ aggregation has been suggested as the critical early inducer, driving the disease progression. However, the factors that promote neurotoxic Aβ aggregation remain elusive. Imaging mass spectrometry (IMS) is a powerful technique to comprehensively elucidate the spatial distribution patterns of lipids, peptides, and proteins in biological tissue sections. In the present study, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS)-based imaging was used on transgenic Alzheimer’s disease mouse (tgArcSwe) brain tissue to investigate the sphingolipid microenvironment of individual Aβ plaques and elucidate plaque-associated sphingolipid alterations. Multivariate data analysis was used to interrogate the IMS data for identifying pathologically relevant, anatomical features based on their lipid chemical profile. This approach revealed sphingolipid species that distinctly located to cortical and hippocampal deposits, whose Aβ identity was further verified using fluorescent amyloid staining and immunohistochemistry. Subsequent multivariate statistical analysis of the spectral data revealed significant localization of gangliosides and ceramides species to Aβ positive plaques, which was accompanied by distinct local reduction of sulfatides. These plaque-associated changes in sphingolipid levels implicate a functional role of sphingolipid metabolism in Aβ plaque pathology and AD pathogenesis. Taken together, the presented data highlight the potential of imaging mass spectrometry as a powerful approach for probing Aβ plaque-associated lipid changes underlying AD pathology. PMID:27984697

  15. Sphingolipid base modifying enzymes in sunflower (Helianthus annuus): cloning and characterization of a C4-hydroxylase gene and a new paralogous Δ8-desaturase gene.

    PubMed

    Moreno-Pérez, Antonio J; Martínez-Force, Enrique; Garcés, Rafael; Salas, Joaquín J

    2011-05-15

    Sphingolipids are components of plant cell membranes that participate in the regulation of important physiological processes. Unlike their animal counterparts, plant sphingolipids are characterized by high levels of base C4-hydroxylation. Moreover, desaturation at the Δ8 position predominates over the Δ4 desaturation typically found in animal sphingolipids. These modifications are due to the action of C4-hydroxylases and Δ8-long chain base desaturases, and they are important for complex sphingolipids finally becoming functional. The long chain bases of sunflower sphingolipids have high levels of hydroxylated and unsaturated moieties. Here, a C4-long chain base hydroxylase was functionally characterized in sunflower plant, an enzyme that could complement the sur2Δ mutation when heterologously expressed in this yeast mutant deficient in hydroxylation. This hydroxylase was ubiquitously expressed in sunflower, with the highest levels found in the developing cotyledons. In addition, we identified a new Δ8-long base chain desaturase gene that displays strong homology to a previously reported desaturase gene. This desaturase was also expressed in yeast and was able to change the long chain base composition of the transformed host. We studied the expression of this desaturase and compared it with that of the other isoform described in sunflower. The desaturase form studied in this paper displayed higher expression levels in developing seeds.

  16. Sphingolipid Metabolites Modulate Dielectric Characteristics of Cells in a Mouse Ovarian Cancer Progression Model

    PubMed Central

    Salmanzadeh, Alireza; Elvington, Elizabeth S.; Roberts, Paul C.

    2013-01-01

    Currently, conventional cancer treatment regimens often rely upon highly toxic chemotherapeutics or target oncogenes that are variably expressed within the heterogeneous cell population of tumors. These challenges highlight the need for novel treatment strategies that 1) are non-toxic yet able to at least partially reverse the aggressive phenotype of the disease to a benign or very slow-growing state, and 2) act on the cells independently of variably expressed biomarkers. Using a label-independent rapid microfluidic cell manipulation strategy known as contactless dielectrophoresis (cDEP), we investigated the effect of non-toxic concentrations of two bioactive sphingolipid metabolites, sphingosine (So), with potential anti-tumor properties, and sphingosine-1-phosphate (S1P), a tumor-promoting metabolite, on the intrinsic electrical properties of early and late stages of mouse ovarian surface epithelial (MOSE) cancer cells. Previously, we demonstrated that electrical properties change as cells progress from a benign early stage to late malignant stages. Here, we demonstrate an association between So treatment and a shift in the bioelectrical characteristics of late stage MOSE (MOSE-L) cells towards a profile similar to that of benign MOSE-E cells. Particularly, the specific membrane capacitance of MOSE-L cells shifted toward that of MOSE-E cells, decreasing from 23.94±2.75 to 16.46±0.62 mF/m2 after So treatment, associated with a decrease in membrane protrusions. In contrast, S1P did not reverse the electrical properties of MOSE-L cells. This work is the first to indicate that treatment with non-toxic doses of So correlates with changes in the electrical properties and surface roughness of cells. It also demonstrates the potential of cDEP to be used as a new, rapid technique for drug efficacy studies, and eventually designing more personalized treatment regimens. PMID:23609351

  17. Annexin II-dependent actin remodelling evoked by hydrogen peroxide requires the metalloproteinase/sphingolipid pathway.

    PubMed

    Cinq-Frais, Christel; Coatrieux, Christelle; Savary, Aude; D'Angelo, Romina; Bernis, Corinne; Salvayre, Robert; Nègre-Salvayre, Anne; Augé, Nathalie

    2015-01-01

    Actin remodeling is a dynamic process associated with cell shape modification occurring during cell cycle and proliferation. Oxidative stress plays a role in actin reorganization via various systems including p38MAPK. Beside, the mitogenic response evoked by hydrogen peroxide (H2O2) in fibroblasts and smooth muscle cells (SMC) involves the metalloproteinase (MMPs)/sphingomyelinase 2 (nSMase2) signaling pathway. The aim of this work was to investigate whether this system plays a role in actin remodeling induced by H2O2. Low H2O2 dose (5µM) rapidly triggered a signaling cascade leading to nSMase2 activation, src and annexin 2 (AnxA2) phosphorylation, and actin remodeling, in fibroblasts and SMC. These events were blocked by pharmacological inhibitors of MMPs (Ro28-2653) and p38MAPK (SB203580), and were lacking in MMP2(-/-) and in nSMase2-mutant (fro) fibroblasts. Likewise, H2O2 was unable to induce actin remodeling in fro and MMP2(-/-) fibroblasts or in cells pretreated with p38MAPK, or MMP inhibitors. Finally we show that nSMase2 activation by H2O2, depends on MMP2 and p38MAPK, and is required for the src-dependent phosphorylation of AnxA2, and actin remodeling. Taken together, these findings indicate for the first time that AnxA2 phosphorylation and actin remodeling evoked by oxidative stress depend on the sphingolipid pathway, via MMP2 and p38MAPK.

  18. Annexin II-dependent actin remodelling evoked by hydrogen peroxide requires the metalloproteinase/sphingolipid pathway

    PubMed Central

    Cinq-Frais, Christel; Coatrieux, Christelle; Savary, Aude; D’Angelo, Romina; Bernis, Corinne; Salvayre, Robert; Nègre-Salvayre, Anne; Augé, Nathalie

    2014-01-01

    Actin remodeling is a dynamic process associated with cell shape modification occurring during cell cycle and proliferation. Oxidative stress plays a role in actin reorganization via various systems including p38MAPK. Beside, the mitogenic response evoked by hydrogen peroxide (H2O2) in fibroblasts and smooth muscle cells (SMC) involves the metalloproteinase (MMPs)/sphingomyelinase 2 (nSMase2) signaling pathway. The aim of this work was to investigate whether this system plays a role in actin remodeling induced by H2O2. Low H2O2 dose (5 µM) rapidly triggered a signaling cascade leading to nSMase2 activation, src and annexin 2 (AnxA2) phosphorylation, and actin remodeling, in fibroblasts and SMC. These events were blocked by pharmacological inhibitors of MMPs (Ro28-2653) and p38MAPK (SB203580), and were lacking in MMP2−/− and in nSMase2-mutant (fro) fibroblasts. Likewise, H2O2 was unable to induce actin remodeling in fro and MMP2−/− fibroblasts or in cells pretreated with p38MAPK, or MMP inhibitors. Finally we show that nSMase2 activation by H2O2, depends on MMP2 and p38MAPK, and is required for the src-dependent phosphorylation of AnxA2, and actin remodeling. Taken together, these findings indicate for the first time that AnxA2 phosphorylation and actin remodeling evoked by oxidative stress depend on the sphingolipid pathway, via MMP2 and p38MAPK. PMID:25574848

  19. MRP- and BCL-2-mediated drug resistance in human SCLC: effects of apoptotic sphingolipids in vitro.

    PubMed

    Khodadadian, M; Leroux, M E; Auzenne, E; Ghosh, S C; Farquhar, D; Evans, R; Spohn, W; Zou, Y; Klostergaard, J

    2009-10-01

    Multidrug-resistance-associated protein (MRP) and BCL-2 contribute to drug resistance expressed in SCLC. To establish whether MRP-mediated drug resistance affects sphingolipid (SL)-induced apoptosis in SCLC, we first examined the human SCLC cell line, UMCC-1, and its MRP over-expressing, drug-resistant subline, UMCC-1/VP. Despite significantly decreased sensitivity to doxorubicin (Dox) and to the etoposide, VP-16, the drug-selected line was essentially equally as sensitive to treatment with exogenous ceramide (Cer), sphingosine (Sp) or dimethyl-sphingosine (DMSP) as the parental line. Next, we observed that high BCL-2-expressing human H69 SCLC cells, that were approximately 160-fold more sensitive to Dox than their combined BCL-2 and MRP-over-expressing (H69AR) counterparts, were only approximately 5-fold more resistant to DMSP. Time-lapse fluorescence microscopy of either UMCC cell line treated with DMSP-Coumarin revealed comparable extents and kinetics of SL uptake, further ruling out MRP-mediated effects on drug uptake. DMSP potentiated the cytotoxic activity of VP-16 and Taxol, but not Dox, in drug-resistant UMCC-1/VP cells. However, this sensitization did not appear to involve DMSP-mediated effects on the function of MRP in drug export; nor did DMSP strongly shift the balance of pro-apoptotic Sps and anti-apoptotic Sp-1-Ps in these cells. We conclude that SL-induced apoptosis markedly overcomes or bypasses MRP-mediated drug resistance relevant to SCLC and may suggest a novel therapeutic approach to chemotherapy for these tumors.

  20. Identification of a New Class of Antifungals Targeting the Synthesis of Fungal Sphingolipids.

    PubMed

    Mor, Visesato; Rella, Antonella; Farnoud, Amir M; Singh, Ashutosh; Munshi, Mansa; Bryan, Arielle; Naseem, Shamoon; Konopka, James B; Ojima, Iwao; Bullesbach, Erika; Ashbaugh, Alan; Linke, Michael J; Cushion, Melanie; Collins, Margaret; Ananthula, Hari Krishna; Sallans, Larry; Desai, Pankaj B; Wiederhold, Nathan P; Fothergill, Annette W; Kirkpatrick, William R; Patterson, Thomas; Wong, Lai Hong; Sinha, Sunita; Giaever, Guri; Nislow, Corey; Flaherty, Patrick; Pan, Xuewen; Cesar, Gabriele Vargas; de Melo Tavares, Patricia; Frases, Susana; Miranda, Kildare; Rodrigues, Marcio L; Luberto, Chiara; Nimrichter, Leonardo; Del Poeta, Maurizio

    2015-06-23

    Recent estimates suggest that >300 million people are afflicted by serious fungal infections worldwide. Current antifungal drugs are static and toxic and/or have a narrow spectrum of activity. Thus, there is an urgent need for the development of new antifungal drugs. The fungal sphingolipid glucosylceramide (GlcCer) is critical in promoting virulence of a variety of human-pathogenic fungi. In this study, we screened a synthetic drug library for compounds that target the synthesis of fungal, but not mammalian, GlcCer and found two compounds [N'-(3-bromo-4-hydroxybenzylidene)-2-methylbenzohydrazide (BHBM) and its derivative, 3-bromo-N'-(3-bromo-4-hydroxybenzylidene) benzohydrazide (D0)] that were highly effective in vitro and in vivo against several pathogenic fungi. BHBM and D0 were well tolerated in animals and are highly synergistic or additive to current antifungals. BHBM and D0 significantly affected fungal cell morphology and resulted in the accumulation of intracellular vesicles. Deep-sequencing analysis of drug-resistant mutants revealed that four protein products, encoded by genes APL5, COS111, MKK1, and STE2, which are involved in vesicular transport and cell cycle progression, are targeted by BHBM. Fungal infections are a significant cause of morbidity and mortality worldwide. Current antifungal drugs suffer from various drawbacks, including toxicity, drug resistance, and narrow spectrum of activity. In this study, we have demonstrated that pharmaceutical inhibition of fungal glucosylceramide presents a new opportunity to treat cryptococcosis and various other fungal infections. In addition to being effective against pathogenic fungi, the compounds discovered in this study were well tolerated by animals and additive to current antifungals. These findings suggest that these drugs might pave the way for the development of a new class of antifungals. Copyright © 2015 Mor et al.

  1. Overexpression of Arabidopsis Ceramide Synthases Differentially Affects Growth, Sphingolipid Metabolism, Programmed Cell Death, and Mycotoxin Resistance1[OPEN

    PubMed Central

    Luttgeharm, Kyle D.; Chen, Ming; Mehra, Amit; Cahoon, Rebecca E.; Markham, Jonathan E.; Cahoon, Edgar B.

    2015-01-01

    Ceramide synthases catalyze an N-acyltransferase reaction using fatty acyl-coenzyme A (CoA) and long-chain base (LCB) substrates to form the sphingolipid ceramide backbone and are targets for inhibition by the mycotoxin fumonisin B1 (FB1). Arabidopsis (Arabidopsis thaliana) contains three genes encoding ceramide synthases with distinct substrate specificities: LONGEVITY ASSURANCE GENE ONE HOMOLOG1 (LOH1; At3g25540)- and LOH3 (At1g19260)-encoded ceramide synthases use very-long-chain fatty acyl-CoA and trihydroxy LCB substrates, and LOH2 (At3g19260)-encoded ceramide synthase uses palmitoyl-CoA and dihydroxy LCB substrates. In this study, complementary DNAs for each gene were overexpressed to determine the role of individual isoforms in physiology and sphingolipid metabolism. Differences were observed in growth resulting from LOH1 and LOH3 overexpression compared with LOH2 overexpression. LOH1- and LOH3-overexpressing plants had enhanced biomass relative to wild-type plants, due in part to increased cell division, suggesting that enhanced synthesis of very-long-chain fatty acid/trihydroxy LCB ceramides promotes cell division and growth. Conversely, LOH2 overexpression resulted in dwarfing. LOH2 overexpression also resulted in the accumulation of sphingolipids with C16 fatty acid/dihydroxy LCB ceramides, constitutive induction of programmed cell death, and accumulation of salicylic acid, closely mimicking phenotypes observed previously in LCB C-4 hydroxylase mutants defective in trihydroxy LCB synthesis. In addition, LOH2- and LOH3-overexpressing plants acquired increased resistance to FB1, whereas LOH1-overexpressing plants showed no increase in FB1 resistance, compared with wild-type plants, indicating that LOH1 ceramide synthase is most strongly inhibited by FB1. Overall, the findings described here demonstrate that overexpression of Arabidopsis ceramide synthases results in strongly divergent physiological and metabolic phenotypes, some of which have significance

  2. Sphingolipid long-chain-base auxotrophs of Saccharomyces cerevisiae: genetics, physiology, and a method for their selection.

    PubMed Central

    Pinto, W J; Srinivasan, B; Shepherd, S; Schmidt, A; Dickson, R C; Lester, R L

    1992-01-01

    A selection method for sphingolipid long-chain-base auxotrophs of Saccharomyces cerevisiae was devised after observing that strains that require a long-chain base for growth become denser when starved for this substance. Genetic analysis of over 60 such strains indicated only two complementation classes, lcb1 and lcb2. Mutant strains from each class grew equally well with 3-ketodihydrosphingosine, erythrodihydrosphingosine or threodihydrosphingosine, or phytosphingosine. Since these metabolites represent the first, second, and last components, respectively, of the long-chain-base biosynthetic pathway, it is likely that the LCB1 and LCB2 genes are involved in the first step of long-chain-base synthesis. The results of long-chain-base starvation in the Lcb- strains suggest that one or more sphingolipids have a vital role in S. cerevisiae. Immediate sequelae of long-chain-base starvation were loss of viability, exacerbated in the presence of alpha-cyclodextrin, and loss of phosphoinositol sphingolipid synthesis but not phosphatidylinositol synthesis. Loss of viability with long-chain-base starvation could be prevented by also blocking either protein or nucleic acid synthesis. Without a long-chain-base, cell division, dry mass accumulation, and protein synthesis continued at a diminished rate and were further inhibited by the detergent Tergitol. The cell density increase induced by long-chain-base starvation is thus explained as a differential loss of cell division and mass accumulation. Long-chain-base starvation in Lcb- S. cerevisiae and inositol starvation of Inos- S. cerevisiae share common features: an increase in cell density and a loss of cell viability overcome by blocking macromolecular synthesis. PMID:1556075

  3. Modifications of Sphingolipid Content Affect Tolerance to Hemibiotrophic and Necrotrophic Pathogens by Modulating Plant Defense Responses in Arabidopsis1[OPEN

    PubMed Central

    Magnin-Robert, Maryline; Le Bourse, Doriane; Markham, Jonathan; Dorey, Stéphan; Clément, Christophe; Baillieul, Fabienne; Dhondt-Cordelier, Sandrine

    2015-01-01

    Sphingolipids are emerging as second messengers in programmed cell death and plant defense mechanisms. However, their role in plant defense is far from being understood, especially against necrotrophic pathogens. Sphingolipidomics and plant defense responses during pathogenic infection were evaluated in the mutant of long-chain base phosphate (LCB-P) lyase, encoded by the dihydrosphingosine-1-phosphate lyase1 (AtDPL1) gene and regulating long-chain base/LCB-P homeostasis. Atdpl1 mutants exhibit tolerance to the necrotrophic fungus Botrytis cinerea but susceptibility to the hemibiotrophic bacterium Pseudomonas syringae pv tomato (Pst). Here, a direct comparison of sphingolipid profiles in Arabidopsis (Arabidopsis thaliana) during infection with pathogens differing in lifestyles is described. In contrast to long-chain bases (dihydrosphingosine [d18:0] and 4,8-sphingadienine [d18:2]), hydroxyceramide and LCB-P (phytosphingosine-1-phosphate [t18:0-P] and 4-hydroxy-8-sphingenine-1-phosphate [t18:1-P]) levels are higher in Atdpl1-1 than in wild-type plants in response to B. cinerea. Following Pst infection, t18:0-P accumulates more strongly in Atdpl1-1 than in wild-type plants. Moreover, d18:0 and t18:0-P appear as key players in Pst- and B. cinerea-induced cell death and reactive oxygen species accumulation. Salicylic acid levels are similar in both types of plants, independent of the pathogen. In addition, salicylic acid-dependent gene expression is similar in both types of B. cinerea-infected plants but is repressed in Atdpl1-1 after treatment with Pst. Infection with both pathogens triggers higher jasmonic acid, jasmonoyl-isoleucine accumulation, and jasmonic acid-dependent gene expression in Atdpl1-1 mutants. Our results demonstrate that sphingolipids play an important role in plant defense, especially toward necrotrophic pathogens, and highlight a novel connection between the jasmonate signaling pathway, cell death, and sphingolipids. PMID:26378098

  4. Ultra-high-performance liquid chromatography electrospray ionization tandem mass spectrometry for accurate analysis of glycerophospholipids and sphingolipids in drug resistance tumor cells.

    PubMed

    Li, Lin; Wang, Linlin; Shangguan, Dihua; Wei, Yanbo; Han, Juanjuan; Xiong, Shaoxiang; Zhao, Zhenwen

    2015-02-13

    Glycerophospholipids and sphingolipids are important signaling molecules which are involved in many physiological and pathological processes. Here we reported an effective method for accurate analysis of these lipids by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The methanol method was adopted for extraction of lipids due to its simplicity and high efficiency. It was found that two subclasses of sphingolipids, sulfatide (ST) and cerebroside (CB), were heat labile, so a decreased temperature in the ion source of MS might be necessary for these compounds analysis. In addition, it was found that the isobaric interferences were commonly existent, for example, the m/z of 16:0/18:1 PC containing two (13)C isotope being identical to that of 16:0/18:0 PC determined by a unit mass resolution mass spectrometer; therefore, a baseline separation of interferential species was required to maintain selectivity and accuracy of analysis. In this work, an ultra-high-performance liquid chromatography (UHPLC)-based method was developed for separation of interferential species. Moreover, in order to deal with the characteristics of different polarity and wide dynamic range of glycerophospholipids and sphingolipids in biological systems, three detecting conditions were combined together for comprehensive and rational analysis of glycerophospholipids and sphingolipids. The method was utilized to profile glycerophospholipids and sphingolipids in drug resistant tumor cells. Our results showed that many lipids were significantly changed in drug resistant tumor cells compared to paired drug sensitive tumor cells. This is a systematic report about the isobaric interferences and heat labile compounds interferences when analyzing glycerophospholipids and sphingolipids by ESI-MS/MS, which aids in ruling out one potential source of systematic error to ensure the accuracy of analysis.

  5. The sphingolipid long-chain base-Pkh1/2-Ypk1/2 signaling pathway regulates eisosome assembly and turnover.

    PubMed

    Luo, Guangzuo; Gruhler, Albrecht; Liu, Ying; Jensen, Ole N; Dickson, Robert C

    2008-04-18

    Eisosomes are recently described fungal structures that play roles in the organization of the plasma membrane and endocytosis. Their major protein components are Pil1 and Lsp1, and previous studies showed that these proteins are phosphorylated by the sphingolipid long-chain base-activated Pkh1 and Pkh2 protein kinases in vitro. We show that Pkh1 and Pkh2 phosphorylate Pil1 and Lsp1 in vivo to produce species B, and that heat stress, which activates Pkh1 and Pkh2, generates a more highly phosphorylated species, C. Cells with low Pkh activity lack species B and C and contain abnormally organized eisosomes. To verify that Pil1 phosphorylation is essential for correct eisosome organization, phosphorylated serine and threonine residues were identified and changed to alanines. A variant Pil1 protein lacking five phosphorylation sites did not form eisosomes during log phase growth, indicating that phosphorylation is critical for eisosome organization. We also found that eisosomes are dynamic structures and disassemble when the Ypk protein kinases, which are activated by the sphingolipid-Pkh signaling pathway, are inactivated or when the sphingolipid signal is pharmacologically blocked with myriocin. We conclude that eisosome formation and turnover are regulated by the sphingolipid-Pkh1/2-Ypk1/2 signaling pathway. These data and previous data showing that endocytosis is regulated by the sphingolipid-Pkh1/2-Ypk1/2 signaling pathway suggest that Pkh1 and -2 respond to changes in membrane sphingolipids and transmit this information to eisosomes via Pil1 phosphorylation. Eisosomes then control endocytosis to align the composition and function of the plasma membrane to match demand.

  6. Induction of cell wall thickening by the antifungal compound dihydromaltophilin disrupts fungal growth and is mediated by sphingolipid biosynthesis.

    PubMed

    Li, Shaojie; Calvo, Ana M; Yuen, Gary Y; Du, Liangcheng; Harris, Steven D

    2009-01-01

    Dihydromaltophilin (heat-stable antifungal factor [HSAF]) is an antifungal metabolite produced in Lysobacter enzymogenes biocontrol strain C3. This compound induces cell wall thickening in Aspergillus nidulans. Here we show that the cell wall thickening is a general response to HSAF in diverse fungal species. In the A. nidulans model, the thickened cell wall negatively affects hyphal growth. Growth of HSAF-pre-treated hyphae failed to resume at hyphal tips with thick cell wall and the actin cable could not re-polarize at the thickened region of the cell wall, even after the treated hyphae were transferred to drug-free medium. Moreover, HSAF-induced cell wall thickening is mediated by sphingolipid synthesis: HSAF failed to induce cell wall thickening in the absence of ceramide synthase BarA and the sphingolipid synthesis inhibitor myriocin was able to suppress HSAF-induced cell wall thickening. The thickened cell wall could be digested by chitinase suggesting that chitin contributes to the HSAF-induced thickening. Furthermore, HSAF treatment activated the transcription of two chitin synthase encoding genes chsB and chsC.

  7. A combined fluorescence spectroscopy, confocal and 2-photon microscopy approach to re-evaluate the properties of sphingolipid domains.

    PubMed

    Pinto, Sandra N; Fernandes, Fábio; Fedorov, Alexander; Futerman, Anthony H; Silva, Liana C; Prieto, Manuel

    2013-09-01

    The aim of this study is to provide further insight about the interplay between important signaling lipids and to characterize the properties of the lipid domains formed by those lipids in membranes containing distinct composition. To this end, we have used a combination of fluorescence spectroscopy, confocal and two-photon microscopy and a stepwise approach to re-evaluate the biophysical properties of sphingolipid domains, particularly lipid rafts and ceramide (Cer)-platforms. By using this strategy we were able to show that, in binary mixtures, sphingolipids (Cer and sphingomyelin, SM) form more tightly packed gel domains than those formed by phospholipids with similar acyl chain length. In more complex lipid mixtures, the interaction between the different lipids is intricate and is strongly dictated by the Cer-to-Chol ratio. The results show that in quaternary phospholipid/SM/Chol/Cer mixtures, Cer forms gel domains that become less packed as Chol is increased. Moreover, the extent of gel phase formation is strongly reduced in these mixtures, even though Cer molar fraction is increased. These results suggest that in biological membranes, lipid domains such as rafts and ceramide platforms, might display distinctive biophysical properties depending on the local lipid composition at the site of the membrane where they are formed, further highlighting the potential role of membrane biophysical properties as an underlying mechanism for mediating specific biological processes.

  8. Role of a new mammalian gene family in the biosynthesis of very long chain fatty acids and sphingolipids.

    PubMed

    Tvrdik, P; Westerberg, R; Silve, S; Asadi, A; Jakobsson, A; Cannon, B; Loison, G; Jacobsson, A

    2000-05-01

    Whereas the physiological significance of microsomal fatty acid elongation is generally appreciated, its molecular nature is poorly understood. Here, we describe tissue-specific regulation of a novel mouse gene family encoding components implicated in the synthesis of very long chain fatty acids. The Ssc1 gene appears to be ubiquitously expressed, whereas Ssc2 and Cig30 show a restricted expression pattern. Their translation products are all integral membrane proteins with five putative transmembrane domains. By complementing the homologous yeast mutants, we found that Ssc1 could rescue normal sphingolipid synthesis in the sur4/elo3 mutant lacking the ability to synthesize cerotic acid (C(26:0)). Similarly, Cig30 reverted the phenotype of the fen1/elo2 mutant that has reduced levels of fatty acids in the C(20)-C(24) range. Further, we show that Ssc1 mRNA levels were markedly decreased in the brains of myelin-deficient mouse mutants known to have very low fatty acid chain elongation activity. Conversely, the dramatic induction of Cig30 expression during brown fat recruitment coincided with elevated elongation activity. Our results strongly implicate this new mammalian gene family in tissue-specific synthesis of very long chain fatty acids and sphingolipids.

  9. Cholesterol- and Sphingolipid-rich Microdomains Are Essential for Microtubule-based Membrane Protrusions Induced by Clostridium difficile Transferase (CDT)*

    PubMed Central

    Schwan, Carsten; Nölke, Thilo; Kruppke, Anna S.; Schubert, Daniel M.; Lang, Alexander E.; Aktories, Klaus

    2011-01-01

    Clostridium difficile toxin (CDT) is a binary actin-ADP-ribosylating toxin that causes depolymerization of the actin cytoskeleton and formation of microtubule-based membrane protrusions, which are suggested to be involved in enhanced bacterial adhesion and colonization of hypervirulent C. difficile strains. Here, we studied the involvement of membrane lipid components of human colon adenocarcinoma (Caco-2) cells in formation of membrane protrusions. Depletion of cholesterol by methyl-β-cyclodextrin inhibited protrusion formation in a concentration-dependent manner but had no major effect on the toxin-catalyzed modification of actin in target cells. Repletion of cholesterol reconstituted formation of protrusions and increased velocity and total amount of protrusion formation. Methyl-β-cyclodextrin had no effect on the CDT-induced changes in the dynamics of microtubules. Formation of membrane protrusions was also inhibited by the cholesterol-binding polyene antibiotic nystatin. Degradation or inhibition of synthesis of sphingolipids by sphingomyelinase and myriocin, respectively, blocked CDT-induced protrusion formation. Benzyl alcohol, which increases membrane fluidity, prevented protrusion formation. CDT-induced membrane protrusions were stained by flotillin-2 and by the fluorescent-labeled lipid raft marker cholera toxin subunit B, which selectively interacts with GM1 ganglioside mainly located in lipid microdomains. The data suggest that formation and especially the initiation of CDT-induced microtubule-based membrane protrusions depend on cholesterol- and sphingolipid-rich lipid microdomains. PMID:21705797

  10. Comprehensive Plasma Metabolomic Analyses of Atherosclerotic Progression Reveal Alterations in Glycerophospholipid and Sphingolipid Metabolism in Apolipoprotein E-deficient Mice

    PubMed Central

    Dang, Vi T.; Huang, Aric; Zhong, Lexy H.; Shi, Yuanyuan; Werstuck, Geoff H.

    2016-01-01

    Atherosclerosis is the major underlying cause of most cardiovascular diseases. Despite recent advances, the molecular mechanisms underlying the pathophysiology of atherogenesis are not clear. In this study, comprehensive plasma metabolomics were used to investigate early-stage atherosclerotic development and progression in chow-fed apolipoprotein E-deficient mice at 5, 10 and 15 weeks of age. Comprehensive plasma metabolomic profiles, based on 4365 detected metabolite features, differentiate atherosclerosis-prone from atherosclerosis-resistant models. Metabolites in the sphingomyelin pathway were significantly altered prior to detectable lesion formation and at all subsequent time-points. The cytidine diphosphate-diacylglycerol pathway was up-regulated during stage I of atherosclerosis, while metabolites in the phosphatidylethanolamine and glycosphingolipid pathways were augmented in mice with stage II lesions. These pathways, involving glycerophospholipid and sphingolipid metabolism, were also significantly affected during the course of atherosclerotic progression. Our findings suggest that distinct plasma metabolomic profiles can differentiate the different stages of atherosclerotic progression. This study reveals that alteration of specific, previously unreported pathways of glycerophospholipid and sphingolipid metabolism are associated with atherosclerosis. The clear difference in the level of several metabolites supports the use of plasma lipid profiling as a diagnostic tool of atherogenesis. PMID:27721472

  11. Fumonisin B1 (FB1) Induces Lamellar Separation and Alters Sphingolipid Metabolism of In Vitro Cultured Hoof Explants

    PubMed Central

    Reisinger, Nicole; Dohnal, Ilse; Nagl, Veronika; Schaumberger, Simone; Schatzmayr, Gerd; Mayer, Elisabeth

    2016-01-01

    One of the most important hoof diseases is laminitis. Yet, the pathology of laminitis is not fully understood. Different bacterial toxins, e.g. endotoxins or exotoxins, seem to play an important role. Additionally, ingestion of mycotoxins, toxic secondary metabolites of fungi, might contribute to the onset of laminitis. In this respect, fumonsins are of special interest since horses are regarded as species most susceptible to this group of mycotoxins. The aim of our study was to investigate the influence of fumonisin B1 (FB1) on primary isolated epidermal and dermal hoof cells, as well as on the lamellar tissue integrity and sphingolipid metabolism of hoof explants in vitro. There was no effect of FB1 at any concentration on dermal or epidermal cells. However, FB1 significantly reduced the separation force of explants after 24 h of incubation. The Sa/So ratio was significantly increased in supernatants of explants incubated with FB1 (2.5–10 µg/mL) after 24 h. Observed effects on Sa/So ratio were linked to significantly increased sphinganine concentrations. Our study showed that FB1 impairs the sphingolipid metabolism of explants and reduces lamellar integrity at non-cytotoxic concentrations. FB1 might, therefore, affect hoof health. Further in vitro and in vivo studies are necessary to elucidate the effects of FB1 on the equine hoof in more detail. PMID:27023602

  12. Identification of Sphingolipid Metabolites That Induce Obesity via Misregulation of Appetite, Caloric Intake and Fat Storage in Drosophila

    PubMed Central

    Walls, Stanley M.; Attle, Steve J.; Brulte, Gregory B.; Walls, Marlena L.; Finley, Kim D.; Chatfield, Dale A.; Herr, Deron R.; Harris, Greg L.

    2013-01-01

    Obesity is defined by excessive lipid accumulation. However, the active mechanistic roles that lipids play in its progression are not understood. Accumulation of ceramide, the metabolic hub of sphingolipid metabolism, has been associated with metabolic syndrome and obesity in humans and model systems. Here, we use Drosophila genetic manipulations to cause accumulation or depletion of ceramide and sphingosine-1-phosphate (S1P) intermediates. Sphingolipidomic profiles were characterized across mutants for various sphingolipid metabolic genes using liquid chromatography electrospray ionization tandem mass spectroscopy. Biochemical assays and microscopy were used to assess classic hallmarks of obesity including elevated fat stores, increased body weight, resistance to starvation induced death, increased adiposity, and fat cell hypertrophy. Multiple behavioral assays were used to assess appetite, caloric intake, meal size and meal frequency. Additionally, we utilized DNA microarrays to profile differential gene expression between these flies, which mapped to changes in lipid metabolic pathways. Our results show that accumulation of ceramides is sufficient to induce obesity phenotypes by two distinct mechanisms: 1) Dihydroceramide (C14:0) and ceramide diene (C14:2) accumulation lowered fat store mobilization by reducing adipokinetic hormone- producing cell functionality and 2) Modulating the S1P: ceramide (C14:1) ratio suppressed postprandial satiety via the hindgut-specific neuropeptide like receptor dNepYr, resulting in caloric intake-dependent obesity. PMID:24339790

  13. Interfacial regulation of acid ceramidase activity. Stimulation of ceramide degradation by lysosomal lipids and sphingolipid activator proteins.

    PubMed

    Linke, T; Wilkening, G; Sadeghlar, F; Mozcall, H; Bernardo, K; Schuchman, E; Sandhoff, K

    2001-02-23

    The lysosomal degradation of ceramide is catalyzed by acid ceramidase and requires sphingolipid activator proteins (SAP) as cofactors in vivo. The aim of this study was to investigate how ceramide is hydrolyzed by acid ceramidase at the water-membrane interface in the presence of sphingolipid activator proteins in a liposomal assay system. The degradation of membrane-bound ceramide was significantly increased both in the absence and presence of SAP-D when anionic lysosomal phospholipids such as bis(monoacylglycero)phosphate, phosphatidylinositol, and dolichol phosphate were incorporated into substrate-bearing liposomes. Higher ceramide degradation rates were observed in vesicles with increased membrane curvature. Dilution assays indicated that acid ceramidase remained bound to the liposomal surface during catalysis. Not only SAP-D, but also SAP-C and SAP-A, were found to be stimulators of ceramide hydrolysis in the presence of anionic phospholipids. This finding was confirmed by cell culture studies, in which SAP-A, -C, and -D reduced the amount of ceramide storage observed in fibroblasts of a patient suffering from prosaposin deficiency. Strong protein-lipid interactions were observed for both SAP-D and acid ceramidase in surface plasmon resonance experiments. Maximum binding of SAP-D and acid ceramidase to lipid bilayers occurred at pH 4.0. Our results demonstrate that anionic, lysosomal lipids are required for efficient hydrolysis of ceramide by acid ceramidase.

  14. Loss of Inositol Phosphorylceramide Sphingolipid Mannosylation Induces Plant Immune Responses and Reduces Cellulose Content in Arabidopsis[OPEN

    PubMed Central

    Ishikawa, Toshiki; Rennie, Emilie A.; Lao, Jeemeng; Yan, Jingwei; Tsai, Alex Yi-Lin; Baidoo, Edward E.K.; Demura, Taku; Kawai-Yamada, Maki

    2016-01-01

    Glycosylinositol phosphorylceramides (GIPCs) are a class of glycosylated sphingolipids found in plants, fungi, and protozoa. These lipids are abundant in the plant plasma membrane, forming ∼25% of total plasma membrane lipids. Little is known about the function of the glycosylated headgroup, but two recent studies have indicated that they play a key role in plant signaling and defense. Here, we show that a member of glycosyltransferase family 64, previously named ECTOPICALLY PARTING CELLS1, is likely a Golgi-localized GIPC-specific mannosyl-transferase, which we renamed GIPC MANNOSYL-TRANSFERASE1 (GMT1). Sphingolipid analysis revealed that the Arabidopsis thaliana gmt1 mutant almost completely lacks mannose-carrying GIPCs. Heterologous expression of GMT1 in Saccharomyces cerevisiae and tobacco (Nicotiana tabacum) cv Bright Yellow 2 resulted in the production of non-native mannosylated GIPCs. gmt1 displays a severe dwarfed phenotype and a constitutive hypersensitive response characterized by elevated salicylic acid and hydrogen peroxide levels, similar to that we previously reported for the Golgi-localized, GIPC-specific, GDP-Man transporter GONST1 (Mortimer et al., 2013). Unexpectedly, we show that gmt1 cell walls have a reduction in cellulose content, although other matrix polysaccharides are unchanged. PMID:27895225

  15. Modulation of the sphingolipid rheostat is involved in paclitaxel resistance of the human prostate cancer cell line PC3-PR.

    PubMed

    Aoyama, Yuka; Sobue, Sayaka; Mizutani, Naoki; Inoue, Chisato; Kawamoto, Yoshiyuki; Nishizawa, Yuji; Ichihara, Masatoshi; Kyogashima, Mamoru; Suzuki, Motoshi; Nozawa, Yoshinoti; Murate, Takashi

    2017-04-29

    Taxoids are anti-cancer drugs frequently used to treat solid tumors, but they are sometimes ineffective and tumors may become resistant to their action. Here, we examined the involvement of sphingolipid metabolic enzymes in paclitaxel (PTX) resistance using a human prostate cancer cell line, PC3, and its PTX-resistant subline, PC3-PR. PTX (20 nM) suppressed cell proliferation and increased various ceramide species in PC3, but not PC3-PR, cells. PC3-PR contained higher S1P levels than did PC3, regardless of PTX treatment. Western blotting revealed that PC3-PR cells expressed higher levels of sphingosine kinase 1 (SPHK1) and glucosylceramide synthase (GCS) but lower levels of acid sphingomyelinase (ASMase) and neutral sphingomyelinase 2 than did PC3 cells. Inhibition of SPHK1 using siRNA or a pharmacological inhibitor decreased S1P levels in PC3-PR cells and inhibited proliferation in the presence or absence of PTX, suggesting that SPHK1 is at least partially responsible for PTX resistance. Similarly, GCS inhibitors (PDMP and PPMP) increased cellular ceramides and suppressed the proliferation of PC3-PR. However, inhibition of proteasome function or histone deacetylase activity increased SMase and ceramide levels and suppressed PC3-PR proliferation. These results suggest that modulation of metabolic enzyme expression and alteration of the sphingolipid rheostat protects cancer cells against PTX. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Transcription factor Ctip2 controls epidermal lipid metabolism and regulates expression of genes involved in sphingolipid biosynthesis during skin development

    PubMed Central

    Wang, Zhixing; Kirkwood, Jay S.; Taylor, Alan W.; Stevens, Jan F.; Leid, Mark; Ganguli-Indra, Gitali; Indra, Arup K.

    2012-01-01

    The stratum corneum is composed of protein-enriched corneocytes embedded in an intercellular matrix of nonpolar lipids organized as lamellar layers and give rise to epidermal permeability barrier (EPB). EPB defects play an important role in the pathophysiology of skin diseases such as eczema. The transcriptional control of skin lipid metabolism is poorly understood. We have discovered that mouse lacking a transcription factor COUP-TF interacting protein 2 (Ctip2) exhibit EPB defects including altered keratinocyte terminal differentiation, delayed skin barrier development and interrupted neutral lipid distribution in the epidermis. We adapted herein a targeted lipidomic approach using mass spectrometry, and have determined that Ctip2−/− mice (germline deletion of Ctip2 gene) display altered composition of major epidermal lipids such as ceramides and sphingomyelins compared to wildtype at different stages of skin development. Interestingly, expressions of several genes involved in skin sphingolipid biosynthesis and metabolism were altered in mutant skin. Ctip2 was found to be recruited to the promoter region of a subset of those genes, suggesting their possible direct regulation by Ctip2. Our results confirm an important role of Ctip2 in regulating skin lipid metabolism and indicate that profiling of epidermal sphingolipid could be useful for designing effective strategies to improve barrier dysfunctions. PMID:23096701

  17. Inhibition of GM3 Synthase Attenuates Neuropathology of Niemann-Pick Disease Type C by Affecting Sphingolipid Metabolism

    PubMed Central

    Lee, Hyun; Lee, Jong Kil; Bae, Yong Chul; Yang, Song Hyun; Okino, Nozomu; Schuchman, Edward H.; Yamashita, Tadashi; Bae, Jae-sung; Jin, Hee Kyung

    2014-01-01

    In several lysosomal storage disorders, including Niemann-Pick disease Type C (NP-C), sphingolipids, including glycosphingolipids, particularly gangliosides, are the predominant storage materials in the brain, raising the possibility that accumulation of these lipids may be involved in the NP-C neurodegenerative process. However, correlation of these accumulations and NP-C neuropathology has not been fully characterized. Here we derived NP-C mice with complete and partial deletion of the Siat9 (encoding GM3 synthase) gene in order to investigate the role of ganglioside in NP-C pathogenesis. According to our results, NPC mice with homozygotic deletion of GM3 synthase exhibited an enhanced neuropathological phenotype and died significantly earlier than NP-C mice. Notably, in contrast to complete depletion, NP-C mice with partial deletion of the GM3 synthase gene showed ameliorated NP-C neuropathology, including motor disability, demyelination, and abnormal accumulation of cholesterol and sphingolipids. These findings indicate the crucial role of GM3 synthesis in the NP-C phenotype and progression of CNS pathologic abnormality, suggesting that well-controlled inhibition of GM3 synthesis could be used as a therapeutic strategy. PMID:24599001

  18. Transient Receptor Potential Canonical 1 (TRPC1) Channels as Regulators of Sphingolipid and VEGF Receptor Expression

    PubMed Central

    Asghar, Muhammad Yasir; Magnusson, Melissa; Kemppainen, Kati; Sukumaran, Pramod; Löf, Christoffer; Pulli, Ilari; Kalhori, Veronica; Törnquist, Kid

    2015-01-01

    The identity of calcium channels in the thyroid is unclear. In human follicular thyroid ML-1 cancer cells, sphingolipid sphingosine 1-phosphate (S1P), through S1P receptors 1 and 3 (S1P1/S1P3), and VEGF receptor 2 (VEGFR2) stimulates migration. We show that human thyroid cells express several forms of transient receptor potential canonical (TRPC) channels, including TRPC1. In TRPC1 knockdown (TRPC1-KD) ML-1 cells, the basal and S1P-evoked invasion and migration was attenuated. Furthermore, the expression of S1P3 and VEGFR2 was significantly down-regulated. Transfecting wild-type ML-1 cells with a nonconducting TRPC1 mutant decreased S1P3 and VEGFR2 expression. In TRPC1-KD cells, receptor-operated calcium entry was decreased. To investigate whether the decreased receptor expression was due to attenuated calcium entry, cells were incubated with the calcium chelator BAPTA-AM (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid). In these cells, and in cells where calmodulin and calmodulin-dependent kinase were blocked pharmacologically, S1P3 and VEGFR2 expression was decreased. In TRPC1-KD cells, both hypoxia-inducible factor 1α expression and the secretion and activity of MMP2 and MMP9 were attenuated, and proliferation was decreased in TRPC1-KD cells. This was due to a prolonged G1 phase of the cell cycle, a significant increase in the expression of the cyclin-dependent kinase inhibitors p21 and p27, and a decrease in the expression of cyclin D2, cyclin D3, and CDK6. Transfecting TRPC1 to TRPC1-KD cells rescued receptor expression, migration, and proliferation. Thus, the expression of S1P3 and VEGFR2 is mediated by a calcium-dependent mechanism. TRPC1 has a crucial role in this process. This regulation is important for the invasion, migration, and proliferation of thyroid cancer cells. PMID:25971967

  19. Age-dependent changes in the sphingolipid composition of CD4+ T cell membranes and immune synapses implicate glucosylceramides in age-related T cell dysfunction

    USDA-ARS?s Scientific Manuscript database

    Sphingolipid (SL4) composition can influence the biophysical properties of cell membranes. Additionally, specific SL modulate signaling pathways involved in proliferation, senescence, and apoptosis. We investigated age-dependent changes in the SL composition of CD4+ T cells, and the impact of these ...

  20. Sphingolipids Containing Very-Long-Chain Fatty Acids Define a Secretory Pathway for Specific Polar Plasma Membrane Protein Targeting in Arabidopsis[W

    PubMed Central

    Markham, Jonathan E.; Molino, Diana; Gissot, Lionel; Bellec, Yannick; Hématy, Kian; Marion, Jessica; Belcram, Katia; Palauqui, Jean-Christophe; Satiat-JeuneMaître, Béatrice; Faure, Jean-Denis

    2011-01-01

    Sphingolipids are a class of structural membrane lipids involved in membrane trafficking and cell polarity. Functional analysis of the ceramide synthase family in Arabidopsis thaliana demonstrates the existence of two activities selective for the length of the acyl chains. Very-long-acyl-chain (C > 18 carbons) but not long-chain sphingolipids are essential for plant development. Reduction of very-long-chain fatty acid sphingolipid levels leads in particular to auxin-dependent inhibition of lateral root emergence that is associated with selective aggregation of the plasma membrane auxin carriers AUX1 and PIN1 in the cytosol. Defective targeting of polar auxin carriers is characterized by specific aggregation of Rab-A2a– and Rab-A1e–labeled early endosomes along the secretory pathway. These aggregates correlate with the accumulation of membrane structures and vesicle fragmentation in the cytosol. In conclusion, sphingolipids with very long acyl chains define a trafficking pathway with specific endomembrane compartments and polar auxin transport protein cargoes. PMID:21666002

  1. Effects of inhibitors of key enzymes of sphingolipid metabolism on insulin-induced glucose uptake and glycogen synthesis in liver cells of old rats.

    PubMed

    Babenko, N A; Kharchenko, V S

    2015-01-01

    Sphingolipids play an important role in the development of insulin resistance. Ceramides are the most potent inhibitors of insulin signal transduction. Ceramides are generated in response to stress stimuli and in old age. In this work, we studied the possible contribution of different pathways of sphingolipid metabolism in age-dependent insulin resistance development in liver cells. Inhibition of key enzymes of sphingolipid synthesis (serine palmitoyl transferase, ceramide synthase) and degradation (neutral and acidic SMases) by means of specific inhibitors (myriocin, fumonisin B1, imipramine, and GW4869) was followed with the reduction of ceramide level and partly improved insulin regulation of glucose metabolism in "old" hepatocytes. Imipramine and GW4869 decreased significantly the acidic and neutral SMase activities, respectively. Treatment of "old" cells with myriocin or fumonisin B1 reduced the elevated in old age ceramide and SM synthesis. Ceramide and SM levels and glucose metabolism regulation by insulin could be improved with concerted action of all tested inhibitors of sphingolipid turnover on hepatocytes. The data demonstrate that not only newly synthesized ceramide and SM but also neutral and acidic SMase-dependent ceramide accumulation plays an important role in development of age-dependent insulin resistance.

  2. Hydroxylation state of fatty acid and long-chain base moieties of sphingolipid determine the sensitivity to growth inhibition due to AUR1 repression in Saccharomyces cerevisiae.

    PubMed

    Tani, Motohiro; Kuge, Osamu

    2012-01-13

    The structures of ceramide found in the yeast Saccharomyces cerevisiae are classified into five groups according to the hydroxylation states of the long-chain base and fatty acid moieties. This diversity is created through the action of enzymes encoded by SUR2, SCS7, and as yet unidentified hydroxylation enzyme(s). Aur1p is an enzyme catalyzing the formation of inositol phosphorylceramide in the yeast, and the defect leads to strong growth inhibition due to accumulation of ceramide and reductions in complex sphingolipid levels. In this study, we found that the deletion of SCS7 results in the enhancement of growth inhibition due to repression of AUR1 expression under the control of a tetracycline-regulatable promoter, whereas the deletion of SUR2 attenuates the growth inhibition. Under AUR1-repressive conditions, SCS7 and SUR2 mutants showed reductions in the complex sphingolipid levels and the accumulation of ceramide, like wild-type cells. On the other hand, the deletion of SCS7 had no effect on the growth inhibition through reductions in the complex sphingolipid levels caused by repression of LIP1 encoding a ceramide synthase subunit. Furthermore, the deletion of SUR2 did not suppress the growth inhibition under LIP1-repressive conditions. Therefore, it is suggested that the deletion of sphingolipid hydroxylases changes the toxicity of ceramide under AUR1-repressive conditions. Copyright © 2011 Elsevier Inc. All rights reserved.

  3. Detailed Structural Characterization of Sphingolipids via 193 nm Ultraviolet Photodissociation and Ultra High Resolution Tandem Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Ryan, Eileen; Nguyen, Catherine Quynh Nhu; Shiea, Christopher; Reid, Gavin E.

    2017-07-01

    Sphingolipids serve not only as components of cellular membranes but also as bioactive mediators of numerous cellular functions. As the biological activities of these lipids are dependent on their structures, and due to the limitations of conventional ion activation methods employed during tandem mass spectrometry (MS/MS), there is a recognized need for the development of improved structure-specific methods for their comprehensive identification and characterization. Here, positive-ionization mode 193 nm ultraviolet photodissociation (UVPD)-MS/MS has been implemented for the detailed structural characterization of lipid species from a range of sphingolipid classes introduced to the mass spectrometer via electrospray ionization as their lithiated or protonated adducts. These include sphingosine d18:1(4E), dihydrosphingosine (sphinganine) d18:0, sphingadiene d18:2(4E,11Z), the isomeric sphingolipids ceramide d18:1(4E)/18:0 and dihydroceramide d18:0/18:1(9Z), ceramide-1-phosphate d18:1(4Z)/16:0, sphingomyelin d18:1(4E)/18:1(9Z) the glycosphingolipids galactosyl ceramide d18:1(4E)/24:1(15Z) and lactosyl ceramide d18:1(4E)/24:0, and several endogenous lipids present within a porcine brain total lipid extract. In addition to the product ions formed by higher energy collision dissociation (HCD), UVPD is shown to yield a series of novel structurally diagnostic product ions resulting from cleavage of both sphingosine carbon-carbon and acyl chain carbon-carbon double bonds for direct localization of site(s) of unsaturation, as well as via diagnostic cleavages of the sphingosine backbone and N-C amide bond linkages. With activation timescales and dissociation efficiencies similar to those found in conventional MS/MS strategies, this approach is therefore a promising new tool in the arsenal of ion activation techniques toward providing complete structural elucidation in automated, high-throughput lipid analysis workflows.

  4. Syringomycin E Inhibition of Saccharomyces cerevisiae: Requirement for Biosynthesis of Sphingolipids with Very-Long-Chain Fatty Acids and Mannose- and Phosphoinositol-Containing Head Groups†

    PubMed Central

    Stock, Stephen D.; Hama, Hiroko; Radding, Jeffrey A.; Young, Debra A.; Takemoto, Jon Y.

    2000-01-01

    Syringomycin E is an antifungal cyclic lipodepsinonapeptide that inhibits the growth of Saccharomyces cerevisiae by interaction with the plasma membrane. A screen conducted to find the yeast genes necessary for its fungicidal action identified two novel syringomycin E response genes, SYR3 and SYR4. A syr3 mutant allele was complemented by ELO2 and ELO3. These genes encode enzymes that catalyze the elongation of sphingolipid very long chain fatty acids. Tetrad analysis showed that SYR3 was ELO2. Strains with deletions of SYR3/ELO2 and ELO3 were resistant to syringomycin E, and lipid analyses of both mutants revealed shortened fatty acid chains and lower levels of sphingolipids. SYR4 was identified by Tn5 inactivation of genomic library plasmids that complemented a syr4 mutant allele. SYR4 was found to be identical to IPT1, which encodes the terminal sphingolipid biosynthetic enzyme, mannosyl-diinositolphosphoryl-ceramide synthase. Deletion Δsyr4/ipt1 strains were viable, were resistant to syringomycin E, did not produce mannosyl-diinositolphosphoryl-ceramide, and accumulated mannosyl-inositolphosphoryl-ceramide. Accumulation of mannosyl-inositolphosphoryl-ceramide was not responsible for resistance since a temperature-sensitive secretory pathway mutant (sec14-3ts) accumulated this sphingolipid and was sensitive to syringomycin E. Finally, Δcsg1/sur1 and Δcsg2 strains defective in the transfer of mannose to inositolphosphoryl-ceramide were resistant to syringomycin E. These findings show that syringomycin E growth inhibition of yeast is promoted by the production of sphingolipids with fully elongated fatty acid chains and the mannosyl and terminal phosphorylinositol moieties of the polar head group. PMID:10770748

  5. Normal phase liquid chromatography coupled to quadrupole time of flight atmospheric pressure chemical ionization mass spectrometry for separation, detection and mass spectrometric profiling of neutral sphingolipids and cholesterol.

    PubMed

    Farwanah, Hany; Wirtz, Jennifer; Kolter, Thomas; Raith, Klaus; Neubert, Reinhard H H; Sandhoff, Konrad

    2009-10-01

    Many lipidomic approaches focus on investigating aspects of sphingolipid metabolism. Special emphasis is put on neutral sphingolipids and cholesterol and their interaction. Such an interest is attributed to the fact that those lipids are altered in a series of serious disorders including various sphingolipidoses. High performance thin-layer chromatography (HPTLC) has become a widely used technique for lipid analysis. However, mass spectrometric profiling is irreplaceable for gaining an overview about the various molecular species within a lipid class. In this work we have developed a sensitive method based on a gradient normal phase high performance liquid chromatography (HPLC) coupled to quadrupole time of flight (QTOF) atmospheric pressure chemical ionization mass spectrometry (APCI-MS) in positive mode, which for the first time enables separation, on-line detection, and mass spectrometric profiling of multiple neutral sphingolipids including ceramide, glucosylceramide, lactosylceramide, globotriaosylceramide, globotetraosylceramide, sphingomyelin as well as cholesterol within less than 15min. An important advantage of the presented HPLC/APCI-MS approach is that the separation pattern emulates the one obtained by an optimized HPTLC method with a multiple stage development. Thus, the lipid classes previously separated and quantified by HPTLC can be easily screened regarding their mass spectrometric profiles by HPLC/APCI-MS. In addition, the selected ionization conditions enable in-source fragmentation providing useful structural information. The methods (HPLC/APCI-MS and the optimized HPTLC) were applied for the analysis of the mentioned lipids in human fibroblasts. This approach is aimed basically at investigators who perform studies based on genetic modifications or treatment with pharmacological agents leading to changes in the biochemical pathways of neutral sphingolipids and cholesterol. In addition, it can be of interest for research on disorders related to

  6. A plasma-membrane E-MAP reveals links of the eisosome with sphingolipid metabolism and endosomal trafficking

    PubMed Central

    Aguilar, Pablo S; Fröhlich, Florian; Rehman, Michael; Shales, Mike; Ulitsky, Igor; Olivera-Couto, Agustina; Braberg, Hannes; Shamir, Ron; Walter, Peter; Mann, Matthias; Ejsing, Christer S; Krogan, Nevan J; Walther, Tobias C

    2011-01-01

    The plasma membrane delimits the cell and controls material and information exchange between itself and the environment. How different plasma-membrane processes are coordinated and how the relative abundance of plasma-membrane lipids and proteins is homeostatically maintained are not yet understood. Here, we used a quantitative genetic interaction map, or E-MAP, to functionally interrogate a set of ~400 genes involved in various aspects of plasma-membrane biology, including endocytosis, signaling, lipid metabolism and eisosome function. From this E-MAP, we derived a set of 57,799 individual interactions between genes functioning in these various processes. Using triplet genetic motif analysis, we identified a new component of the eisosome, Eis1, and linked the poorly characterized gene EMP70 to endocytic and eisosome function. Finally, we implicated Rom2, a GDP/GTP exchange factor for Rho1 and Rho2, in the regulation of sphingolipid metabolism. PMID:20526336

  7. A plasma-membrane E-MAP reveals links of the eisosome with sphingolipid metabolism and endosomal trafficking.

    PubMed

    Aguilar, Pablo S; Fröhlich, Florian; Rehman, Michael; Shales, Mike; Ulitsky, Igor; Olivera-Couto, Agustina; Braberg, Hannes; Shamir, Ron; Walter, Peter; Mann, Matthias; Ejsing, Christer S; Krogan, Nevan J; Walther, Tobias C

    2010-07-01

    The plasma membrane delimits the cell and controls material and information exchange between itself and the environment. How different plasma-membrane processes are coordinated and how the relative abundance of plasma-membrane lipids and proteins is homeostatically maintained are not yet understood. Here, we used a quantitative genetic interaction map, or E-MAP, to functionally interrogate a set of approximately 400 genes involved in various aspects of plasma-membrane biology, including endocytosis, signaling, lipid metabolism and eisosome function. From this E-MAP, we derived a set of 57,799 individual interactions between genes functioning in these various processes. Using triplet genetic motif analysis, we identified a new component of the eisosome, Eis1, and linked the poorly characterized gene EMP70 to endocytic and eisosome function. Finally, we implicated Rom2, a GDP/GTP exchange factor for Rho1 and Rho2, in the regulation of sphingolipid metabolism.

  8. The Synthetic Amphipathic Peptidomimetic LTX109 Is a Potent Fungicide That Disturbs Plasma Membrane Integrity in a Sphingolipid Dependent Manner

    PubMed Central

    Larsen, Camilla Eggert; Folkesson, Anders; Regenberg, Birgitte

    2013-01-01

    The peptidomimetic LTX109 (arginine-tertbutyl tryptophan-arginine-phenylethan) was previously shown to have antibacterial properties. Here, we investigated the activity of this novel antimicrobial peptidomimetic on the yeast Saccharomyces cerevisiae. We found that LTX109 was an efficient fungicide that killed all viable cells in an exponentially growing population as well as a large proportion of cells in biofilm formed on an abiotic surface. LTX109 had similar killing kinetics to the membrane-permeabilizing fungicide amphotericin B, which led us to investigate the ability of LTX109 to disrupt plasma membrane integrity. S. cerevisiae cells exposed to a high concentration of LTX109 showed rapid release of potassium and amino acids, suggesting that LTX109 acted by destabilizing the plasma membrane. This was supported by the finding that cells were permeable to the fluorescent nucleic acid stain SYTOX Green after a few minutes of LTX109 treatment. We screened a haploid S. cerevisiae gene deletion library for mutants resistant to LTX109 to uncover potential molecular targets. Eight genes conferred LTX109 resistance when deleted and six were involved in the sphingolipid biosynthetic pathway (SUR1, SUR2, SKN1, IPT1, FEN1 and ORM2). The involvement of all of these genes in the biosynthetic pathway for the fungal-specific lipids mannosylinositol phosphorylceramide (MIPC) and mannosyl di-(inositol phosphoryl) ceramide (M(IP)2C) suggested that these lipids were essential for LTX109 sensitivity. Our observations are consistent with a model in which LTX109 kills S. cerevisiae by nonspecific destabilization of the plasma membrane through direct or indirect interaction with the sphingolipids. PMID:23874964

  9. The yeast magmas ortholog pam16 has an essential function in fermentative growth that involves sphingolipid metabolism.

    PubMed

    Short, Mary K; Hallett, Joshua P; Tar, Krisztina; Dange, Thomas; Schmidt, Marion; Moir, Robyn; Willis, Ian M; Jubinsky, Paul T

    2012-01-01

    Magmas is a growth factor responsive gene encoding an essential mitochondrial protein in mammalian cells. Pam16, the Magmas ortholog in Saccharomyces cerevisiae, is a component of the presequence translocase-associated motor. A temperature-sensitive allele (pam16-I61N) was used to query an array of non-essential gene-deletion strains for synthetic genetic interactions. The pam16-I61N mutation at ambient temperature caused synthetic lethal or sick phenotypes with genes involved in lipid metabolism, perixosome synthesis, histone deacetylation and mitochondrial protein import. The gene deletion array was also screened for suppressors of the pam16-I61N growth defect to identify compensatory pathways. Five suppressor genes were identified (SUR4, ISC1, IPT1, SKN1, and FEN1) and all are involved in sphingolipid metabolism. pam16-I61N cells cultured in glucose at non-permissive temperatures resulted in rapid growth inhibition and G1 cell cycle arrest, but cell viability was maintained. Altered mitochondria morphology, reduced peroxisome induction in glycerol/ethanol and oleate, and changes in the levels of several sphingolipids including C18 alpha-hydroxy-phytoceramide, were also observed in the temperature sensitive strain. Deletion of SUR4, the strongest suppressor, reversed the temperature sensitive fermentative growth defect, the morphological changes and the elevated levels of C18 alpha-hydroxy phytoceramide in pam16-I61N. Deletion of the other four suppressor genes had similar effects on C18 alpha-hydroxy-phytoceramide levels and restored proliferation to the pam16-I61N strain. In addition, pam16-I61N inhibited respiratory growth, likely by reducing cardiolipin, which is essential for mitochondrial function. Our results suggest that the pleiotropic effects caused by impaired Pam16/Magmas function are mediated in part by changes in lipid metabolism.

  10. Plasma sphingolipids are biomarkers of metabolic syndrome in non-human primates maintained on a Western-style diet.

    PubMed

    Brozinick, J T; Hawkins, E; Hoang Bui, H; Kuo, M-S; Tan, B; Kievit, P; Grove, K

    2013-08-01

    The intake of a Western diet enriched in animal fat has been shown to be a major risk factor for Type 2 diabetes and obesity. Previous rodent studies have indicated that these conditions may be triggered by the accumulation of the sphingolipid ceramide in insulin-sensitive tissues. However, data are lacking in this regard from both humans and non-human primates. Here we have investigated the relationship between plasma ceramides and metabolic syndrome in Rhesus macaques fed a high-fat and high-fructose (HFFD) 'western' diet. We investigated this relationship in cohorts of monkeys fed a HFFD for a period of 8 months to 5 years. Animals were classified as control, pre-diabetic or diabetic based on fasting plasma parameters and insulin sensitivity. HFFD treatment produced significant increases in body weight and body fat and also resulted in a decline in insulin sensitivity. In parallel to the reduction in insulin sensitivity, significant increases in both plasma ceramide and dihydroceramide levels were observed, which further increased as animals progressed to the diabetic state. Plasma levels of the rare sphingolipid C18:0 deoxysphinganine, a marker of increased metabolic flux through serine palmitoyl transferase (SPT), were also elevated in both pre- and diabetic animals. Furthermore, plasma serine levels were significantly elevated in diabetic monkeys, which may indicate a shift in SPT substrate selectivity from serine to alanine or glycine. In contrast, branch chain amino acids were unchanged in pre-diabetic non-human primates, and only plasma valine levels were elevated in diabetic animals. Together, these data indicate that HFFD induces de novo synthesis of ceramides in non-human primates, and that increased production of plasma ceramides is significantly correlated with the decline in insulin sensitivity.

  11. The Yeast Magmas Ortholog Pam16 Has an Essential Function in Fermentative Growth That Involves Sphingolipid Metabolism

    PubMed Central

    Short, Mary K.; Hallett, Joshua P.; Tar, Krisztina; Dange, Thomas; Schmidt, Marion; Moir, Robyn; Willis, Ian M.; Jubinsky, Paul T.

    2012-01-01

    Magmas is a growth factor responsive gene encoding an essential mitochondrial protein in mammalian cells. Pam16, the Magmas ortholog in Saccharomyces cerevisiae, is a component of the presequence translocase-associated motor. A temperature-sensitive allele (pam16-I61N) was used to query an array of non-essential gene-deletion strains for synthetic genetic interactions. The pam16-I61N mutation at ambient temperature caused synthetic lethal or sick phenotypes with genes involved in lipid metabolism, perixosome synthesis, histone deacetylation and mitochondrial protein import. The gene deletion array was also screened for suppressors of the pam16-I61N growth defect to identify compensatory pathways. Five suppressor genes were identified (SUR4, ISC1, IPT1, SKN1, and FEN1) and all are involved in sphingolipid metabolism. pam16-I61N cells cultured in glucose at non-permissive temperatures resulted in rapid growth inhibition and G1 cell cycle arrest, but cell viability was maintained. Altered mitochondria morphology, reduced peroxisome induction in glycerol/ethanol and oleate, and changes in the levels of several sphingolipids including C18 alpha-hydroxy-phytoceramide, were also observed in the temperature sensitive strain. Deletion of SUR4, the strongest suppressor, reversed the temperature sensitive fermentative growth defect, the morphological changes and the elevated levels of C18 alpha-hydroxy phytoceramide in pam16-I61N. Deletion of the other four suppressor genes had similar effects on C18 alpha-hydroxy-phytoceramide levels and restored proliferation to the pam16-I61N strain. In addition, pam16-I61N inhibited respiratory growth, likely by reducing cardiolipin, which is essential for mitochondrial function. Our results suggest that the pleiotropic effects caused by impaired Pam16/Magmas function are mediated in part by changes in lipid metabolism. PMID:22808036

  12. The synthetic amphipathic peptidomimetic LTX109 is a potent fungicide that disturbs plasma membrane integrity in a sphingolipid dependent manner.

    PubMed

    Bojsen, Rasmus; Torbensen, Rasmus; Larsen, Camilla Eggert; Folkesson, Anders; Regenberg, Birgitte

    2013-01-01

    The peptidomimetic LTX109 (arginine-tertbutyl tryptophan-arginine-phenylethan) was previously shown to have antibacterial properties. Here, we investigated the activity of this novel antimicrobial peptidomimetic on the yeast Saccharomyces cerevisiae. We found that LTX109 was an efficient fungicide that killed all viable cells in an exponentially growing population as well as a large proportion of cells in biofilm formed on an abiotic surface. LTX109 had similar killing kinetics to the membrane-permeabilizing fungicide amphotericin B, which led us to investigate the ability of LTX109 to disrupt plasma membrane integrity. S. cerevisiae cells exposed to a high concentration of LTX109 showed rapid release of potassium and amino acids, suggesting that LTX109 acted by destabilizing the plasma membrane. This was supported by the finding that cells were permeable to the fluorescent nucleic acid stain SYTOX Green after a few minutes of LTX109 treatment. We screened a haploid S. cerevisiae gene deletion library for mutants resistant to LTX109 to uncover potential molecular targets. Eight genes conferred LTX109 resistance when deleted and six were involved in the sphingolipid biosynthetic pathway (SUR1, SUR2, SKN1, IPT1, FEN1 and ORM2). The involvement of all of these genes in the biosynthetic pathway for the fungal-specific lipids mannosylinositol phosphorylceramide (MIPC) and mannosyl di-(inositol phosphoryl) ceramide (M(IP)2C) suggested that these lipids were essential for LTX109 sensitivity. Our observations are consistent with a model in which LTX109 kills S. cerevisiae by nonspecific destabilization of the plasma membrane through direct or indirect interaction with the sphingolipids.

  13. Differential Sphingolipid and Phospholipid Profiles in Alcohol and Nicotine-Derived Nitrosamine Ketone (NNK) Associated White Matter Degeneration

    PubMed Central

    Yalcin, Emine B.; Nunez, Kavin; Tong, Ming; de la Monte, Suzanne M.

    2015-01-01

    Background Alcohol-mediated neurodegeneration is associated with white matter (WM) atrophy due to targeting of myelin and oligodendrocytes. However, variability in disease severity suggests co-factors contribute to WM degeneration. We examined the potential co-factor role of the tobacco-specific nitrosamine, nicotine-derived nitrosamine ketone (NNK), since smoking causes WM atrophy and most heavy drinkers consume tobacco products. Methods This 8-week study of Long Evans rats had 4 treatment groups: control; NNK-2 mg/kg, 3×/wk in Wks 3–8; ethanol (chronic-26% caloric + binge-2 g/kg, 3×/week in Wks 7–8); and ethanol+NNK. Exposure effects on WM lipid biochemical profiles and in situ distributions were examined using matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) and tandem mass spectrometry. Results NNK mainly caused WM fiber degeneration and fiber loss, ethanol caused demyelination, and dual exposures had additive effects. Ethanol and ethanol+NNK decreased WM (including corpus callosum) and/or gray matter (hypothalamus, cortex, medial temporal) levels of several phosphatidylserine (PS), phosphatidylinositol (PI) and sphingolipid (sulfatide; ST) species, while NNK increased or had minimal effect on these lipids. Ethanol+NNK had broader and larger inhibitory effects on phospholipids and sulfatides than ethanol or NNK alone. Principle component analysis clustered control with NNK, and ethanol with ethanol+NNK groups, highlighting the independent ethanol-rather than NNK-driven responses. Conclusion Chronic ethanol exposures decreased several phospholipid and sphingolipid species in brain, while concomitant NNK exposures exacerbated these effects. These findings support our hypothesis that tobacco smoking is a pathogenic co-factor in alcohol-mediated WM degeneration. PMID:26756797

  14. Ceramide sphingolipid signaling mediates Tumor Necrosis Factor (TNF)-dependent toxicity via caspase signaling in dopaminergic neurons

    PubMed Central

    2012-01-01

    Background Dopaminergic (DA) neurons in the ventral midbrain selectively degenerate in Parkinson’s disease (PD) in part because their oxidative environment in the substantia nigra (SN) may render them vulnerable to neuroinflammatory stimuli. Chronic inhibition of soluble Tumor Necrosis Factor (TNF) with dominant-negative TNF inhibitors protects DA neurons in rat models of parkinsonism, yet the molecular mechanisms and pathway(s) that mediate TNF toxicity remain(s) to be clearly identified. Here we investigated the contribution of ceramide sphingolipid signaling in TNF-dependent toxicity. Results Ceramide dose-dependently reduced the viability of DA neuroblastoma cells and primary DA neurons and pharmacological inhibition of sphingomyelinases (SMases) with three different inhibitors during TNF treatment afforded significant neuroprotection by attenuating increased endoplasmic reticulum (ER) stress, loss of mitochondrial membrane potential, caspase-3 activation and decreases in Akt phosphorylation. Using lipidomics mass spectrometry we confirmed that TNF treatment not only promotes generation of ceramide, but also leads to accumulation of several atypical deoxy-sphingoid bases (DSBs). Exposure of DA neuroblastoma cells to atypical DSBs in the micromolar range reduced cell viability and inhibited neurite outgrowth and branching in primary DA neurons, suggesting that TNF-induced de novo synthesis of atypical DSBs may be a secondary mechanism involved in mediating its neurotoxicity in DA neurons. Conclusions We conclude that TNF/TNFR1-dependent activation of SMases generates ceramide and sphingolipid species that promote degeneration and caspase-dependent cell death of DA neurons. Ceramide and atypical DSBs may represent novel drug targets for development of neuroprotective strategies that can delay or attenuate the progressive loss of nigral DA neurons in patients with PD. PMID:22973882

  15. Evaluation of the change in sphingolipids in the human multiple myeloma cell line U266 and gastric cancer cell line MGC-803 treated with arsenic trioxide.

    PubMed

    Zou, Jianhua; Ma, Xiaoqiong; Zhang, Guangji; Shen, Li; Zhou, Liting; Yu, Yu; Zhu, Fanfan; Chen, Zhe

    2015-11-01

    Arsenic trioxide (As2O3) has been found to display anticancer activity against many types of tumors and has been developed into an anticancer drug in clinical treatments. Sphingolipids are membrane lipids that participate in many signal transduction pathways. In this paper, the changes in sphingolipids of the human multiple myeloma cell line U266 and the gastric cancer cell line MGC-803 treated with arsenic trioxide were investigated using an HPLC-ESI-MS/MS method. Analytes were separated by an XBridge BEH C8 column used for Cer, HexCer, LacCer and SM chromatographic separation, and a Capcell PAK MG II C18 column was used for Sph, dhSph, S1P and dhS1P chromatographic separation and gradient elution with acetonitrile-water containing 0.1% formic acid as a mobile phase. A tandem mass spectrometer QTrap in SRM mode was employed in combination with RPLC as a detector for quantitative analysis. The ceramide/sphingolipid internal standard (IS) mixture was used to quantify the levels of sphingolipids. The distributions of sphingolipids were found to be different in the human multiple myeloma cell line U266 and the gastric cancer cell line MGC-803. Ceramide (Cer), hexosylceramide (HexCer) and dihexosylceramide (Hex2Cer) levels in U266 cell line are higher than those in MGC-803 cell line. Additionally, sphingomyelin (SM), sphingosine-1-phosphate (S1P) and sphinganine-1-phosphate (dhS1P) levels in the MGC-803 cell line are higher than those in the U266 cell line. When treated with arsenic trioxide (1-5μM iAs(III)(As(III) ions)), the levels of Hex2Cer in the human multiple myeloma cell line U266 decreased, and the levels of S1P and dhS1P in the human gastric cancer cell line MGC-803 decreased. The decrease of Hex2Cer, S1P and dhS1P in the human multiple myeloma cell line U266 and gastric cancer cell line MGC-803 were observed when the concentration of iAs(III) is 1.0μM. Therefore, arsenic trioxide exhibits anti-cancer activity by altering the sphingolipid pathway in the

  16. Prosaposin Deficiency and Saposin B Deficiency (Activator-Deficient Metachromatic Leukodystrophy): Report on Two Patients Detected by Analysis of Urinary Sphingolipids and Carrying Novel PSAP Gene Mutations

    PubMed Central

    Kuchař, Ladislav; Ledvinová, Jana; Hřebíček, Martin; Myšková, Helena; Dvořáková, Lenka; Berná, Linda; Chrastina, Petr; Asfaw, Befekadu; Elleder, Milan; Petermöller, Margret; Mayrhofer, Heidi; Staudt, Martin; Krägeloh-Mann, Ingeborg; Paton, Barbara C; Harzer, Klaus

    2009-01-01

    Prosaposin deficiency (pSap-d) and saposin B deficiency (SapB-d) are both lipid storage disorders caused by mutations in the PSAP gene that codes for the 65–70 kDa prosaposin protein, which is the precursor for four sphingolipid activator proteins, saposins A–D. We report on two new patients with PSAP gene defects; one, with pSap-d, who had a severe neurovisceral dystrophy and died as a neonate, and the other with SapB-d, who presented with a metachromatic leukodystrophy-like disorder but had normal arylsulfatase activity. Screening for urinary sphingolipids was crucial to the diagnosis of both patients, with electrospray ionization tandem mass spectrometry also providing quantification. The pSap-d patient is the first case with this condition where urinary sphingolipids have been investigated. Multiple sphingolipids were elevated, with globotriaosylceramide showing the greatest increase. Both patients had novel mutations in the PSAP gene. The pSap-d patient was homozygous for a splice-acceptor site mutation two bases upstream of exon 10. This mutation led to a premature stop codon and yielded low levels of transcript. The SapB-d patient was a compound heterozygote with a splice-acceptor site variant exclusively affecting the SapB domain on one allele, and a 2 bp deletion leading to a null, that is, pSap-d mutation, on the other allele. Phenotypically, pSap-d is a relatively uniform disease of the neonate, whereas SapB-d is heterogeneous with a spectrum similar to that in metachromatic leukodystrophy. The possible existence of genotypes and phenotypes intermediate between those of pSap-d and the single saposin deficiencies is speculated. © 2009 Wiley-Liss, Inc. PMID:19267410

  17. Requirement of a specific group of sphingolipid-metabolizing enzyme for growth of yeast Saccharomyces cerevisiae under impaired metabolism of glycerophospholipids.

    PubMed

    Tani, Motohiro; Kuge, Osamu

    2010-10-01

    Sphingolipids play critical roles in many physiologically important events in yeast Saccharomyces cerevisiae. In this study, we screened for yeast mutants showing high sensitivity to Aureobasidin A, an inhibitor of inositol phosphorylceramide synthase, and found that a lack of SAC1 encoding phosphoinositides phosphatase causes high sensitivity to the inhibitor. Double mutation analysis involving the SAC1 and non-essential sphingolipid-metabolizing enzyme genes revealed that csg1Δ, csg2Δ, ipt1Δ or scs7Δ causes synthetic lethality with deletion of SAC1. As previously reported, SAC1-repressed cells exhibited a reduced cellular phosphatidylserine (PS) level, and overexpression of PSS1 encoding PS synthase complemented the growth defects of scs7Δ, csg1Δ and ipt1Δ cells under SAC1-repressive conditions. Furthermore, repression of PSS1 expression resulted in synthetic growth defect with the deletion of CSG1, IPT1 or SCS7. The growth defects of scs7Δ, csg1Δ and ipt1Δ cells under SAC1- or PSS1-repressive conditions were also complemented by overexpression of Arf-GAP AGE1, which encodes a protein related to membrane trafficking. Under SAC1-repressive conditions, scs7Δ, csg1Δ and ipt1Δ cells showed defects in vacuolar morphology, which were complemented by overexpression of each of PSS1 and AGE1. These results suggested that a specific group of sphingolipid-metabolizing enzyme is required for yeast cell growth under impaired metabolism of glycerophospholipids.

  18. Psychosine-triggered endomitosis is modulated by membrane sphingolipids through regulation of phosphoinositide 4,5-bisphosphate production at the cleavage furrow.

    PubMed

    Watanabe, Hiroshi; Okahara, Kyohei; Naito-Matsui, Yuko; Abe, Mitsuhiro; Go, Shinji; Inokuchi, Jinichi; Okazaki, Toshiro; Kobayashi, Toshihide; Kozutsumi, Yasunori; Oka, Shogo; Takematsu, Hiromu

    2016-07-01

    Endomitosis is a special type of mitosis in which only cytokinesis-the final step of the cell division cycle-is defective, resulting in polyploid cells. Although endomitosis is biologically important, its regulatory aspects remain elusive. Psychosine, a lysogalactosylceramide, prevents proper cytokinesis when supplemented to proliferating cells. Cytokinetic inhibition by psychosine does not inhibit genome duplication. Consequently cells undergo multiple rounds of endomitotic cell cycles, resulting in the formation of giant multiploid cells. Here we successfully quantified psychosine-triggered multiploid cell formation, showing that membrane sphingolipids ratios modulate psychosine-triggered polyploidy in Namalwa cells. Among enzymes that experimentally remodel cellular sphingolipids, overexpression of glucosylceramide synthase to biosynthesize glycosylsphingolipids (GSLs) and neutral sphingomyelinase 2 to hydrolyze sphingomyelin (SM) additively enhanced psychosine-triggered multiploidy; almost all of the cells became polyploid. In the presence of psychosine, Namalwa cells showed attenuated cell surface SM clustering and suppression of phosphatidylinositol 4,5-bisphosphate production at the cleavage furrow, both important processes for cytokinesis. Depending on the sphingolipid balance between GSLs and SM, Namalwa cells could be effectively converted to viable multiploid cells with psychosine. © 2016 Watanabe et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  19. Psychosine-triggered endomitosis is modulated by membrane sphingolipids through regulation of phosphoinositide 4,5-bisphosphate production at the cleavage furrow

    PubMed Central

    Watanabe, Hiroshi; Okahara, Kyohei; Naito-Matsui, Yuko; Abe, Mitsuhiro; Go, Shinji; Inokuchi, Jinichi; Okazaki, Toshiro; Kobayashi, Toshihide; Kozutsumi, Yasunori; Oka, Shogo; Takematsu, Hiromu

    2016-01-01

    Endomitosis is a special type of mitosis in which only cytokinesis—the final step of the cell division cycle—is defective, resulting in polyploid cells. Although endomitosis is biologically important, its regulatory aspects remain elusive. Psychosine, a lysogalactosylceramide, prevents proper cytokinesis when supplemented to proliferating cells. Cytokinetic inhibition by psychosine does not inhibit genome duplication. Consequently cells undergo multiple rounds of endomitotic cell cycles, resulting in the formation of giant multiploid cells. Here we successfully quantified psychosine-triggered multiploid cell formation, showing that membrane sphingolipids ratios modulate psychosine-triggered polyploidy in Namalwa cells. Among enzymes that experimentally remodel cellular sphingolipids, overexpression of glucosylceramide synthase to biosynthesize glycosylsphingolipids (GSLs) and neutral sphingomyelinase 2 to hydrolyze sphingomyelin (SM) additively enhanced psychosine-triggered multiploidy; almost all of the cells became polyploid. In the presence of psychosine, Namalwa cells showed attenuated cell surface SM clustering and suppression of phosphatidylinositol 4,5-bisphosphate production at the cleavage furrow, both important processes for cytokinesis. Depending on the sphingolipid balance between GSLs and SM, Namalwa cells could be effectively converted to viable multiploid cells with psychosine. PMID:27170180

  20. HPTLC-MALDI MS for (glyco)sphingolipid multiplexing in tissues and blood: A promising strategy for biomarker discovery and clinical applications.

    PubMed

    Torretta, Enrica; Fania, Chiara; Vasso, Michele; Gelfi, Cecilia

    2016-07-01

    Sphingolipids have hydrophilic and hydrophobic properties, different saturation and combination of the oligosaccharide chains and mass homology of species located in a narrow m/z region hampering their recognition. To target sphingolipids for diagnostic purposes, standardized methods for lipid extraction, quali- and quantitative assessments are required. In this study, HPTLC-MALDI MS was adopted to establish sphingolipid and glycosphingolipid profiles in muscle, brain and serum to create a database of molecules to be searched in the preclinical and clinical investigations. Specific protocols for lipid extraction were set up based on the characteristics of the tissue or/and fluids; this approach maximizes the HPTLC-MALDI MS analytical throughput both for lipids extracted in organic and aqueous phase. This study indicates that alkaline hydrolysis is necessary for the detection of low abundant species such as Gb3Cer and ceramides in serum and Gb4Cer, CerP and HexCer in muscle tissue. The high hydrophobicity of ceramides has been overcome by the development of HPTLC plate in chloroform:methanol/50:3.5, which increases the number and the intensity of low abundant Cer species. MS/MS analysis has been conducted directly on HPTLC plate allowing the molecular recognition; furthermore a dataset of spectra was acquired to create a database for future profiling of these molecules.

  1. Structure and Nanomechanics of Model Membranes by Atomic Force Microscopy and Spectroscopy: Insights into the Role of Cholesterol and Sphingolipids

    PubMed Central

    Gumí-Audenis, Berta; Costa, Luca; Carlá, Francesco; Comin, Fabio; Sanz, Fausto; Giannotti, Marina I.

    2016-01-01

    Biological membranes mediate several biological processes that are directly associated with their physical properties but sometimes difficult to evaluate. Supported lipid bilayers (SLBs) are model systems widely used to characterize the structure of biological membranes. Cholesterol (Chol) plays an essential role in the modulation of membrane physical properties. It directly influences the order and mechanical stability of the lipid bilayers, and it is known to laterally segregate in rafts in the outer leaflet of the membrane together with sphingolipids (SLs). Atomic force microscope (AFM) is a powerful tool as it is capable to sense and apply forces with high accuracy, with distance and force resolution at the nanoscale, and in a controlled environment. AFM-based force spectroscopy (AFM-FS) has become a crucial technique to study the nanomechanical stability of SLBs by controlling the liquid media and the temperature variations. In this contribution, we review recent AFM and AFM-FS studies on the effect of Chol on the morphology and mechanical properties of model SLBs, including complex bilayers containing SLs. We also introduce a promising combination of AFM and X-ray (XR) techniques that allows for in situ characterization of dynamic processes, providing structural, morphological, and nanomechanical information. PMID:27999368

  2. Structure and Nanomechanics of Model Membranes by Atomic Force Microscopy and Spectroscopy: Insights into the Role of Cholesterol and Sphingolipids.

    PubMed

    Gumí-Audenis, Berta; Costa, Luca; Carlá, Francesco; Comin, Fabio; Sanz, Fausto; Giannotti, Marina I

    2016-12-19

    Biological membranes mediate several biological processes that are directly associated with their physical properties but sometimes difficult to evaluate. Supported lipid bilayers (SLBs) are model systems widely used to characterize the structure of biological membranes. Cholesterol (Chol) plays an essential role in the modulation of membrane physical properties. It directly influences the order and mechanical stability of the lipid bilayers, and it is known to laterally segregate in rafts in the outer leaflet of the membrane together with sphingolipids (SLs). Atomic force microscope (AFM) is a powerful tool as it is capable to sense and apply forces with high accuracy, with distance and force resolution at the nanoscale, and in a controlled environment. AFM-based force spectroscopy (AFM-FS) has become a crucial technique to study the nanomechanical stability of SLBs by controlling the liquid media and the temperature variations. In this contribution, we review recent AFM and AFM-FS studies on the effect of Chol on the morphology and mechanical properties of model SLBs, including complex bilayers containing SLs. We also introduce a promising combination of AFM and X-ray (XR) techniques that allows for in situ characterization of dynamic processes, providing structural, morphological, and nanomechanical information.

  3. Sphingolipid metabolites selectively elicit increases in nuclear calcium concentration in cell suspension cultures and in isolated nuclei of tobacco.

    PubMed

    Xiong, Tou Cheu; Coursol, Sylvie; Grat, Sabine; Ranjeva, Raoul; Mazars, Christian

    2008-01-01

    Sphingolipids are known to interfere with calcium-based signalling pathways. Here we report that these compounds modulate nuclear calcium signalling in tobacco BY-2 cells. Nuclear protein kinase activity phosphorylated endogenous sphingoid long-chain bases (LCBs), suggesting that LCBs are actively metabolized in the nucleus of tobacco BY-2 cells. The Delta4-unsaturated LCB D-erythro-sphingosine and the saturated LCB D-ribo-phytosphingosine elicited increases in free calcium in the nucleus in a dose-dependent and structure-related manner. However, neither sphingosine-1-phosphate nor C2-ceramide was able to stimulate nuclear calcium changes. N-,N-Dimethyl-D-erythro-sphingosine, a structural analogue of D-erythro-sphingosine, was the most efficient LCB so far tested in eliciting nuclear calcium changes both in intact tobacco BY-2 cells and in isolated nuclei. TRP channel inhibitors prevent the effect of DMS, suggesting that LCBs may activate TRP-like channels located on the inner nuclear membrane Collectively, the obtained data show that nuclei respond to LCBs on their own independently of the cytosolic compartment.

  4. Chlamydia trachomatis Intercepts Golgi-Derived Sphingolipids through a Rab14-Mediated Transport Required for Bacterial Development and Replication

    PubMed Central

    Capmany, Anahí; Damiani, María Teresa

    2010-01-01

    Chlamydia trachomatis are obligate intracellular bacteria that survive and replicate in a bacterial-modified phagosome called inclusion. As other intracellular parasites, these bacteria subvert the phagocytic pathway to avoid degradation in phagolysosomes and exploit trafficking pathways to acquire both energy and nutrients essential for their survival. Rabs are host proteins that control intracellular vesicular trafficking. Rab14, a Golgi-related Rab, controls Golgi to endosomes transport. Since Chlamydia establish a close relationship with the Golgi apparatus, the recruitment and participation of Rab14 on inclusion development and bacteria growth were analyzed. Time course analysis revealed that Rab14 associated with inclusions by 10 h post infection and was maintained throughout the entire developmental cycle. The recruitment was bacterial protein synthesis-dependent but independent of microtubules and Golgi integrity. Overexpression of Rab14 dominant negative mutants delayed inclusion enlargement, and impaired bacteria replication as determined by IFU. Silencing of Rab14 by siRNA also decreased bacteria multiplication and infectivity. By electron microscopy, aberrant bacteria were observed in cells overexpressing the cytosolic negative Rab14 mutant. Our results showed that Rab14 facilitates the delivery of sphingolipids required for bacterial development and replication from the Golgi to chlamydial inclusions. Novel anti-chlamydial therapies could be developed based on the knowledge of how bacteria subvert host vesicular transport events through Rabs manipulation. PMID:21124879

  5. Long-chain bases of sphingolipids are transported into cells via the acyl-CoA synthetases

    PubMed Central

    Narita, Tomomi; Naganuma, Tatsuro; Sase, Yurie; Kihara, Akio

    2016-01-01

    Transport of dietary lipids into small-intestinal epithelial cells is pathologically and nutritionally important. However, lipid uptake remains an almost unexplored research area. Although we know that long-chain bases (LCBs), constituents of sphingolipids, can enter into cells efficiently, the molecular mechanism of LCB uptake is completely unclear. Here, we found that the yeast acyl-CoA synthetases (ACSs) Faa1 and Faa4 are redundantly involved in LCB uptake. In addition to fatty acid-activating activity, transporter activity toward long-chain fatty acids (LCFAs) has been suggested for ACSs. Both LCB and LCFA transports were largely impaired in faa1Δ faa4Δ cells. Furthermore, LCB and LCFA uptakes were mutually competitive. However, the energy dependency was different for their transports. Sodium azide/2-deoxy-D-glucose treatment inhibited import of LCFA but not that of LCB. Furthermore, the ATP-AMP motif mutation FAA1 S271A largely impaired the metabolic activity and LCFA uptake, while leaving LCB import unaffected. These results indicate that only LCFA transport requires ATP. Since ACSs do not metabolize LCBs as substrates, Faa1 and Faa4 are likely directly involved in LCB transport. Furthermore, we revealed that ACSs are also involved in LCB transport in mammalian cells. Thus, our findings provide strong support for the hypothesis that ACSs directly transport LCFAs. PMID:27136724

  6. The roles of bioactive sphingolipids in resveratrol-induced apoptosis in HL60: acute myeloid leukemia cells.

    PubMed

    Cakir, Zeynep; Saydam, Guray; Sahin, Fahri; Baran, Yusuf

    2011-02-01

    Acute promyelocytic leukemia results from a translocation between 15 and 17 chromosomes that produce PML/RARα fusion protein. PML/RARα inhibits differentiation of myeloid precursor cells at stem cell level. Resveratrol is a phytoalexin that exerts cytotoxic effects on cancer cells. Ceramides have crucial roles in cell growth, proliferation, differentiation, drug resistance, and apoptosis. In this study, we examined the possible cytotoxic effects of resveratrol on acute myeloid leukemia cells and determined the roles of ceramide-metabolizing genes in resveratrol-induced apoptosis, in addition to investigating the possibility of increasing the sensitivity of HL60 cells to resveratrol by manipulating sphingolipids. Cytotoxic effects of resveratrol, C8:ceramide, PDMP, and SK-1 inhibitor were determined by XTT cell proliferation assay. Changes in caspase-3 enzyme activity and mitochondrial membrane potential (MMP) were measured using caspase-3 colorimetric assay and JC-1 MMP detection kit. Expression levels of ceramide-metabolizing genes were examined by RT-PCR. The results revealed that manipulations of ceramide metabolism toward generation or accumulation of apoptotic ceramides increased apoptotic effects of resveratrol in HL60 cells, synergistically. More importantly, gene expression analyses revealed that resveratrol-induced apoptosis via increasing expression levels of ceramide-generating genes and decreasing expression levels of antiapoptotic sphingosine kinase-1 and glucosylceramide synthase genes. These results showed for the first time that increasing intracellular levels of ceramides by biochemical approaches has also increased sensitivity of HL60 cells to resveratrol. We also showed that resveratrol induces apoptosis through manipulating ceramide-metabolizing genes that resulted in the accumulation of ceramides in HL60 cells.

  7. Bedside Stereomicroscopy of Fabry Kidney Biopsies: An Easily Available Method for Diagnosis and Assessment of Sphingolipid Deposits.

    PubMed

    Svarstad, Einar; Leh, Sabine; Skrunes, Rannveig; Kampevold Larsen, Kristin; Eikrem, Øystein; Tøndel, Camilla

    2017-08-26

    A previous case report found stereomicroscopic changes typical for Fabry disease in a kidney biopsy. This case series evaluates an expanded diagnostic capacity of the method. Bedside stereomicroscopy was performed in a cross-sectional prospective study of 31 consecutive enzyme-treated or treatment-naïve male (n = 14) and female Fabry disease patients. The burden of glomerular storage material was scored semiquantitatively on a visual analog scale (range 0-3) and a blinded comparison was done with a reference histologic method. Significant correlations (p < 0.001) were found between the stereomicroscopic scoring of glomerular characteristic white storage material and the amount of podocyte globotriaosylceramide (Gb3) deposits scored by standardized light microscopy. The bedside method correctly identified the variability of podocyte Gb3 accumulation after 10 years of identical agalsidase therapy in 2 brothers aged 24 and 27 years, and also identified tubular cell deposits. Stereomicroscopy correctly verified the absence of sphingolipid deposits in the biopsy of a female index patient with a genetic variant of unknown significance, and the diagnosis of Fabry disease was finally discarded. Bedside stereomicroscopy of kidney biopsies is an easily available, low-cost microscopy method handled by the clinician. The method carries a high diagnostic sensitivity for Fabry disease, reducing the risk of misdiagnosis in previously unknown cases. An expanded yield of the method is suggested, including the grading of the podocyte Gb3 burden and assessment of effectiveness of enzyme replacement therapy. We recommend the method as complementary to current standard histologic evaluation of Fabry kidney biopsies. © 2017 S. Karger AG, Basel.

  8. Lack of ceramide generation and altered sphingolipid composition are associated with drug resistance in human ovarian carcinoma cells

    PubMed Central

    Prinetti, Alessandro; Millimaggi, Danilo; D'Ascenzo, Sandra; Clarkson, Matilda; Bettiga, Arianna; Chigorno, Vanna; Sonnino, Sandro; Pavan, Antonio; Dolo, Vincenza

    2005-01-01

    PTX (Paclitaxel®) is an antimitotic agent used in the treatment of a number of major solid tumours, particularly in breast and ovarian cancer. This study was undertaken to gain insight into the molecular alterations producing PTX resistance in ovarian cancer. PTX treatment is able to induce apoptosis in the human ovarian carcinoma cell line, CABA I. PTX-induced apoptosis in CABA I cells was accompanied by an increase in the cellular Cer (ceramide) levels and a decrease in the sphingomyelin levels, due to the activation of sphingomyelinases. The inhibition of acid sphingomyelinase decreased PTX-induced apoptosis. Under the same experimental conditions, PTX had no effect on Cer and sphingomyelin levels in the stable PTX-resistant ovarian carcinoma cell line, CABA-PTX. The acquisition of the PTX-resistant phenotype is accompanied by unique alterations in the complex sphingolipid pattern found on lipid extraction. In the drug-resistant cell line, the levels of sphingomyelin and neutral glycosphingolipids were unchanged compared with the drug-sensitive cell line. The ganglioside pattern in CABA I cells is more complex compared with that of CABA-PTX cells. Specifically, we found that the total ganglioside content in CABA-PTX cells was approximately half of that in CABA I cells, and GM3 ganglioside content was remarkably higher in the drug-resistant cell line. Taken together our findings indicate that: i) Cer generated by acid sphingomyelinase is involved in PTX-induced apoptosis in ovarian carcinoma cells, and PTX-resistant cells are characterized by their lack of increased Cer upon drug treatment, ii) PTX resistance might be correlated with an alteration in metabolic Cer patterns specifically affecting cellular ganglioside composition. PMID:16356169

  9. Interference with distinct steps of sphingolipid synthesis and signaling attenuates proliferation of U87MG glioma cells

    PubMed Central

    Bernhart, Eva; Damm, Sabine; Wintersperger, Andrea; Nusshold, Christoph; Brunner, Anna Martina; Plastira, Ioanna; Rechberger, Gerald; Reicher, Helga; Wadsack, Christian; Zimmer, Andreas; Malle, Ernst; Sattler, Wolfgang

    2015-01-01

    Glioblastoma is the most common malignant brain tumor, which, despite combined radio- and chemotherapy, recurs and is invariably fatal for affected patients. Members of the sphingolipid (SL) family are potent effectors of glioma cell proliferation. In particular sphingosine-1-phosphate (S1P) and the corresponding G protein-coupled S1P receptors transmit proliferative signals to glioma cells. To investigate the contribution to glioma cell proliferation we inhibited the first step of de novo SL synthesis in p53wt and p53mut glioma cells, and interfered with S1P signaling specifically in p53wt U87MG cells. Subunit silencing (RNAi) or pharmacological antagonism (using myriocin) of serine palmitoyltransferase (SPT; catalyzing the first committed step of SL biosynthesis) reduced proliferation of p53wt but not p53mut GBM cells. In U87MG cells these observations were accompanied by decreased ceramide, sphingomyelin, and S1P content. Inhibition of SPT upregulated p53 and p21 expression and induced an increase in early and late apoptotic U87MG cells. Exogenously added S1P (complexed to physiological carriers) increased U87MG proliferation. In line, silencing of individual members of the S1P receptor family decreased U87MG proliferation. Silencing and pharmacological inhibition of the ATP-dependent cassette transporter A1 (ABCA1) that facilitates S1P efflux in astrocytes attenuated U87MG growth. Glyburide-mediated inhibition of ABCA1 resulted in intracellular accumulation of S1P raising the possibility that ABCA1 promotes S1P efflux in U87MG glioma cells thereby contributing to inside-out signaling. Our findings indicate that de novo SL synthesis, S1P receptor-mediated signaling, and ABCA1-mediated S1P efflux could provide pharmacological targets to interfere with glioma cell proliferation. PMID:26002572

  10. Apical sorting of bovine enteropeptidase does not involve detergent-resistant association with sphingolipid-cholesterol rafts.

    PubMed

    Zheng, X; Lu, D; Sadler, J E

    1999-01-15

    Enteropeptidase is a heterodimeric type II membrane protein of the brush border of duodenal enterocytes. In this location, enteropeptidase cleaves and activates trypsinogen, thereby initiating the activation of other intestinal digestive enzymes. Recombinant bovine enteropeptidase was sorted directly to the apical surface of polarized Madin-Darby canine kidney cells. Replacement of the cytoplasmic and signal anchor domains with a cleavable signal peptide (mutant proenteropeptidase lacking the amino-terminal signal anchor domain (dSA-BEK)) caused apical secretion. The additional amino-terminal deletion of a mucin-like domain (HL-BEK) resulted in secretion both apically and basolaterally. Further deletion of the noncatalytic heavy chain (L-BEK) resulted in apical secretion. Thus enteropeptidase appears to have at least three distinct sorting signals as follows: the light chain (L-BEK) directs apical sorting, addition of most of the heavy chain (HL-BEK) inhibits apical sorting, and addition of the mucin-like domain (dSA-BEK) restores apical sorting. Inhibition of N-linked glycosylation with tunicamycin or disruption of microtubules with colchicine caused L-BEK to be secreted equally into apical and basolateral compartments, whereas brefeldin A caused basolateral secretion of L-BEK. Full-length BEK was not found in detergent-resistant raft domains of Madin-Darby canine kidney cells or baby hamster kidney cells. These results suggest apical sorting of enteropeptidase depends on N-linked glycosylation of the serine protease domain and an amino-terminal segment that includes an O-glycosylated mucin-like domain and three potential N-glycosylation sites. In contrast to many apically targeted proteins, enteropeptidase does not form detergent-resistant associations with sphingolipid-cholesterol rafts.

  11. Genetic, biochemical, and transcriptional responses of Saccharomyces cerevisiae to the novel immunomodulator FTY720 largely mimic those of the natural sphingolipid phytosphingosine.

    PubMed

    Welsch, Carole A; Roth, Lukas W A; Goetschy, Jean François; Movva, N Rao

    2004-08-27

    Sphingolipids are signaling molecules that influence diverse cellular functions from control of the cell cycle to degradation of plasma membrane proteins. The synthetic sphingolipid-like compound FTY720 is an immunomodulating agent in clinical trials for transplant graft maintenance. In this report, we compare the effects of the natural yeast sphingolipid phytosphingosine with FTY720 in Saccharomyces cerevisiae. We show that the multicopy suppressor genes that induce growth resistance to FTY720 also confer resistance to growth-inhibitory concentrations of phytosphingosine. In addition, mutants for ubiquitination pathway proteins are shown to be resistant to the growth-inhibiting effect of both FTY720 and phytosphingosine. We observe fewer similarities between sphingosine and FTY720 than between FTY720 and phytosphingosine as revealed by genetic studies. Yeast cells lacking the specific sphingosine kinase LCB4 are sensitive to phytosphingosine and FTY720 but resistant to sphingosine, suggesting that FTY720 and phytosphingosine have a more related mechanism of action. Gene expression profile comparisons of sensitive and resistant yeast cells exposed to FTY720 and phytosphingosine highlight a number of similarities. In response to treatment with these compounds, approximately 77% of the genes that are regulated >2-fold by FTY720 also respond to phytosphingosine in the same direction in the parent strain. In addition, a close inspection of TAT1 and TAT2 transporters following exposure to phytosphingosine indicates that TAT1 protein is degraded in a similar fashion upon treatment with FTY720 and phytosphingosine. There were differences, however, with respect to the TAT2 protein level and the expression profiles of a subset of genes. The genetic, transcriptional, and biochemical data together indicate that FTY720 and phytosphingosine influence similar pathways in yeast cells. These findings offer further insights into the physiological pathways influenced by these

  12. Expression of the bacterial type III effector DspA/E in Saccharomyces cerevisiae down-regulates the sphingolipid biosynthetic pathway leading to growth arrest.

    PubMed

    Siamer, Sabrina; Guillas, Isabelle; Shimobayashi, Mitsugu; Kunz, Caroline; Hall, Michael N; Barny, Marie-Anne

    2014-06-27

    Erwinia amylovora, the bacterium responsible for fire blight, relies on a type III secretion system and a single injected effector, DspA/E, to induce disease in host plants. DspA/E belongs to the widespread AvrE family of type III effectors that suppress plant defense responses and promote bacterial growth following infection. Ectopic expression of DspA/E in plant or in Saccharomyces cerevisiae is toxic, indicating that DspA/E likely targets a cellular process conserved between yeast and plant. To unravel the mode of action of DspA/E, we screened the Euroscarf S. cerevisiae library for mutants resistant to DspA/E-induced growth arrest. The most resistant mutants (Δsur4, Δfen1, Δipt1, Δskn1, Δcsg1, Δcsg2, Δorm1, and Δorm2) were impaired in the sphingolipid biosynthetic pathway. Exogenously supplied sphingolipid precursors such as the long chain bases (LCBs) phytosphingosine and dihydrosphingosine also suppressed the DspA/E-induced yeast growth defect. Expression of DspA/E in yeast down-regulated LCB biosynthesis and induced a rapid decrease in LCB levels, indicating that serine palmitoyltransferase (SPT), the first and rate-limiting enzyme of the sphingolipid biosynthetic pathway, was repressed. SPT down-regulation was mediated by dephosphorylation and activation of Orm proteins that negatively regulate SPT. A Δcdc55 mutation affecting Cdc55-PP2A protein phosphatase activity prevented Orm dephosphorylation and suppressed DspA/E-induced growth arrest. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. FTY720/fingolimod increases NPC1 and NPC2 expression and reduces cholesterol and sphingolipid accumulation in Niemann-Pick type C mutant fibroblasts.

    PubMed

    Newton, Jason; Hait, Nitai C; Maceyka, Michael; Colaco, Alexandria; Maczis, Melissa; Wassif, Christopher A; Cougnoux, Antony; Porter, Forbes D; Milstien, Sheldon; Platt, Nicholas; Platt, Frances M; Spiegel, Sarah

    2017-04-01

    Niemann-Pick type C (NPC) disease is a fatal neurodegenerative disorder caused by mutations in NPC1 or NPC2 with decreased functions leading to lysosomal accumulation of cholesterol and sphingolipids. FTY720/fingolimod, used for treatment of multiple sclerosis, is phosphorylated by nuclear sphingosine kinase 2, and its active phosphorylated form (FTY720-P) is an inhibitor of class I histone deacetylases. In this study, administration of clinically relevant doses of FTY720 to mice increased expression of NPC1 and -2 in brain and liver and decreased cholesterol in an SphK2-dependent manner. FTY720 greatly increased expression of NPC1 and -2 in human NPC1 mutant fibroblasts that correlated with formation of FTY720-P and significantly reduced the accumulation of cholesterol and glycosphingolipids. In agreement with this finding, FTY720 pretreatment of human NPC1 mutant fibroblasts restored transport of the cholera toxin B subunit, which binds ganglioside GM1, to the Golgi apparatus. Together, these findings suggest that FTY720 administration can ameliorate cholesterol and sphingolipid storage and trafficking defects in NPC1 mutant fibroblasts. Because neurodegeneration is the main clinical feature of NPC disease, and FTY720 accumulates in the CNS and has several advantages over available histone deacetylase inhibitors now in clinical trials, our work provides a potential opportunity for treatment of this incurable disease.-Newton, J., Hait, N. C., Maceyka, M., Colaco, A., Maczis, M., Wassif, C. A., Cougnoux, A., Porter, F. D., Milstien, S., Platt, N., Platt, F. M., Spiegel, S. FTY720/fingolimod increases NPC1 and NPC2 expression and reduces cholesterol and sphingolipid accumulation in Niemann-Pick type C mutant fibroblasts.

  14. Imaging Mass Spectrometry Reveals Acyl-Chain- and Region-Specific Sphingolipid Metabolism in the Kidneys of Sphingomyelin Synthase 2-Deficient Mice.

    PubMed

    Sugimoto, Masayuki; Wakabayashi, Masato; Shimizu, Yoichi; Yoshioka, Takeshi; Higashino, Kenichi; Numata, Yoshito; Okuda, Tomohiko; Zhao, Songji; Sakai, Shota; Igarashi, Yasuyuki; Kuge, Yuji

    2016-01-01

    Obesity was reported to cause kidney injury by excessive accumulation of sphingolipids such as sphingomyelin and ceramide. Sphingomyelin synthase 2 (SMS2) is an important enzyme for hepatic sphingolipid homeostasis and its dysfunction is considered to result in fatty liver disease. The expression of SMS2 is also high in the kidneys. However, the contribution of SMS2 on renal sphingolipid metabolism remains unclear. Imaging mass spectrometry is a powerful tool to visualize the distribution and provide quantitative data on lipids in tissue sections. Thus, in this study, we analyzed the effects of SMS2 deficiency on the distribution and concentration of sphingomyelins in the liver and kidneys of mice fed with a normal-diet or a high-fat-diet using imaging mass spectrometry and liquid chromatography/electrospray ionization-tandem mass spectrometry. Our study revealed that high-fat-diet increased C18-C22 sphingomyelins, but decreased C24-sphingomyelins, in the liver and kidneys of wild-type mice. By contrast, SMS2 deficiency decreased C18-C24 sphingomyelins in the liver. Although a similar trend was observed in the whole-kidneys, the effects were minor. Interestingly, imaging mass spectrometry revealed that sphingomyelin localization was specific to each acyl-chain length in the kidneys. Further, SMS2 deficiency mainly decreased C22-sphingomyelin in the renal medulla and C24-sphingomyelins in the renal cortex. Thus, imaging mass spectrometry can provide visual assessment of the contribution of SMS2 on acyl-chain- and region-specific sphingomyelin metabolism in the kidneys.

  15. Imaging Mass Spectrometry Reveals Acyl-Chain- and Region-Specific Sphingolipid Metabolism in the Kidneys of Sphingomyelin Synthase 2-Deficient Mice

    PubMed Central

    Sugimoto, Masayuki; Wakabayashi, Masato; Shimizu, Yoichi; Yoshioka, Takeshi; Higashino, Kenichi; Numata, Yoshito; Okuda, Tomohiko; Zhao, Songji; Sakai, Shota; Igarashi, Yasuyuki; Kuge, Yuji

    2016-01-01

    Obesity was reported to cause kidney injury by excessive accumulation of sphingolipids such as sphingomyelin and ceramide. Sphingomyelin synthase 2 (SMS2) is an important enzyme for hepatic sphingolipid homeostasis and its dysfunction is considered to result in fatty liver disease. The expression of SMS2 is also high in the kidneys. However, the contribution of SMS2 on renal sphingolipid metabolism remains unclear. Imaging mass spectrometry is a powerful tool to visualize the distribution and provide quantitative data on lipids in tissue sections. Thus, in this study, we analyzed the effects of SMS2 deficiency on the distribution and concentration of sphingomyelins in the liver and kidneys of mice fed with a normal-diet or a high-fat-diet using imaging mass spectrometry and liquid chromatography/electrospray ionization-tandem mass spectrometry. Our study revealed that high-fat-diet increased C18–C22 sphingomyelins, but decreased C24-sphingomyelins, in the liver and kidneys of wild-type mice. By contrast, SMS2 deficiency decreased C18–C24 sphingomyelins in the liver. Although a similar trend was observed in the whole-kidneys, the effects were minor. Interestingly, imaging mass spectrometry revealed that sphingomyelin localization was specific to each acyl-chain length in the kidneys. Further, SMS2 deficiency mainly decreased C22-sphingomyelin in the renal medulla and C24-sphingomyelins in the renal cortex. Thus, imaging mass spectrometry can provide visual assessment of the contribution of SMS2 on acyl-chain- and region-specific sphingomyelin metabolism in the kidneys. PMID:27010944

  16. The antifungal Aureobasidin A and an analogue are active against the protozoan parasite Toxoplasma gondii but do not inhibit sphingolipid biosynthesis.

    PubMed

    Alqaisi, A Q I; Mbekeani, A J; Llorens, M Bassas; Elhammer, A P; Denny, P W

    2017-05-10

    Toxoplasma gondii is an obligate intracellular protozoan parasite of the phylum Apicomplexa, and toxoplasmosis is an important disease of both humans and economically important animals. With a limited array of drugs available there is a need to identify new therapeutic compounds. Aureobasidin A (AbA) is an antifungal that targets the essential inositol phosphorylceramide (IPC, sphingolipid) synthase in pathogenic fungi. This natural cyclic depsipeptide also inhibits Toxoplasma proliforation, with the protozoan IPC synthase orthologue proposed as the target. The data presented here show that neither AbA nor an analogue (Compound 20), target the protozoan IPC synthase orthologue or total parasite sphingolipid synthesis. However, further analyses confirm that AbA exhibits significant activity against the proliferative tachyzoite form of Toxoplasma, and Compound 20, whilst effective, has reduced efficacy. This difference was more evident on analyses of the direct effect of these compounds against isolated Toxoplasma, indicating that AbA is rapidly microbicidal. Importantly, the possibility of targeting the encysted, bradyzoite, form of the parasite with AbA and Compound 20 was demonstrated, indicating that this class of compounds may provide the basis for the first effective treatment for chronic toxoplasmosis.

  17. Functional and phylogenetic evidence of a bacterial origin for the first enzyme in sphingolipid biosynthesis in a phylum of eukaryotic protozoan parasites.

    PubMed

    Mina, John G; Thye, Julie K; Alqaisi, Amjed Q I; Bird, Louise E; Dods, Robert H; Grøftehauge, Morten K; Mosely, Jackie A; Pratt, Steven; Shams-Eldin, Hosam; Schwarz, Ralph T; Pohl, Ehmke; Denny, Paul W

    2017-07-21

    Toxoplasma gondii is an obligate, intracellular eukaryotic apicomplexan protozoan parasite that can cause fetal damage and abortion in both animals and humans. Sphingolipids are essential and ubiquitous components of eukaryotic membranes that are both synthesized and scavenged by the Apicomplexa. Here we report the identification, isolation, and analyses of the Toxoplasma serine palmitoyltransferase, an enzyme catalyzing the first and rate-limiting step in sphingolipid biosynthesis: the condensation of serine and palmitoyl-CoA. In all eukaryotes analyzed to date, serine palmitoyltransferase is a highly conserved heterodimeric enzyme complex. However, biochemical and structural analyses demonstrated the apicomplexan orthologue to be a functional, homodimeric serine palmitoyltransferase localized to the endoplasmic reticulum. Furthermore, phylogenetic studies indicated that it was evolutionarily related to the prokaryotic serine palmitoyltransferase, identified in the Sphingomonadaceae as a soluble homodimeric enzyme. Therefore this enzyme, conserved throughout the Apicomplexa, is likely to have been obtained via lateral gene transfer from a prokaryote. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Effects of stereochemistry, saturation, and hydrocarbon chain length on the ability of synthetic constrained azacyclic sphingolipids to trigger nutrient transporter down-regulation, vacuolation, and cell death.

    PubMed

    Perryman, Michael S; Tessier, Jérémie; Wiher, Timothy; O'Donoghue, Heather; McCracken, Alison N; Kim, Seong M; Nguyen, Dean G; Simitian, Grigor S; Viana, Matheus; Rafelski, Susanne; Edinger, Aimee L; Hanessian, Stephen

    2016-09-15

    Constrained analogs containing a 2-hydroxymethylpyrrolidine core of the natural sphingolipids sphingosine, sphinganine, N,N-dimethylsphingosine and N-acetyl variants of sphingosine and sphinganine (C2-ceramide and dihydro-C2-ceramide) were synthesized and evaluated for their ability to down-regulate nutrient transporter proteins and trigger cytoplasmic vacuolation in mammalian cells. In cancer cells, the disruptions in intracellular trafficking produced by these sphingolipids lead to cancer cell death by starvation. Structure activity studies were conducted by varying the length of the hydrocarbon chain, the degree of unsaturation and the presence or absence of an aryl moiety on the appended chains, and stereochemistry at two stereogenic centers. In general, cytotoxicity was positively correlated with nutrient transporter down-regulation and vacuolation. This study was intended to identify structural and functional features in lead compounds that best contribute to potency, and to develop chemical biology tools that could be used to isolate the different protein targets responsible for nutrient transporter loss and cytoplasmic vacuolation. A molecule that produces maximal vacuolation and transporter loss is expected to have the maximal anti-cancer activity and would be a lead compound.

  19. The effect of the bioactive sphingolipids S1P and C1P on multipotent stromal cells--new opportunities in regenerative medicine.

    PubMed

    Marycz, Krzysztof; Śmieszek, Agnieszka; Jeleń, Marta; Chrząstek, Klaudia; Grzesiak, Jakub; Meissner, Justyna

    2015-09-01

    Sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P) belong to a family of bioactive sphingolipids that act as important extracellular signaling molecules and chemoattractants. This study investigated the influence of S1P and C1P on the morphology, proliferation activity and osteogenic properties of rat multipotent stromal cells derived from bone marrow (BMSCs) and subcutaneous adipose tissue (ASCs). We show that S1P and C1P can influence mesenchymal stem cells (MSCs), each in a different manner. S1P stimulation promoted the formation of cellular aggregates of BMSCs and ASCs, while C1P had an effect on the regular growth pattern and expanded intercellular connections, thereby increasing the proliferative activity. Although osteogenic differentiation of MSCs was enhanced by the addition of S1P, the effectiveness of osteoblast differentiation was more evident in BMSCs, particularly when biochemical and molecular marker levels were considered. The results of the functional osteogenic differentiation assay, which includes an evaluation of the efficiency of extracellular matrix mineralization (SEM-EDX), revealed the formation of numerous mineral aggregates in BMSC cultures stimulated with S1P. Our data demonstrated that in an appropriate combination, the bioactive sphingolipids S1P and C1P may find wide application in regenerative medicine, particularly in bone regeneration with the use of MSCs.

  20. Long N-acyl fatty acids on sphingolipids are responsible for miscibility with phospholipids to form liquid-ordered phase.

    PubMed

    Quinn, Peter J

    2009-10-01

    :phospholipid at 25 degrees C with pure phospholipid in gel phase and 42:58 mole ratio at 65 degrees C when the phospholipid was in the fluid phase. The results are discussed with reference to the role of the length of the N-acyl substituent of the sphingolipids in formation of complexes with phospholipids.

  1. Feeding a Modified Fish Diet to Bottlenose Dolphins Leads to an Increase in Serum Adiponectin and Sphingolipids

    PubMed Central

    Sobolesky, Philip M.; Harrell, Tyler S.; Parry, Celeste; Venn-Watson, Stephanie; Janech, Michael G.

    2016-01-01

    Feeding a modified fish diet has been suggested to improve insulin sensitivity in bottlenose dolphins; however, insulin sensitivity was not directly measured. Since demonstrating an improvement in insulin sensitivity is technically difficult in dolphins, we postulated that directional changes in the hormone axis: fibroblast growth factor 21 (FGF21)/Adiponectin/Ceramide (Cer), could provide further support to this hypothesis. We measured 2-h post-prandial serum FGF21, total adiponectin, percent unmodified adiponectin, ceramide, and sphingosine levels from dolphins fed a diet rich in heptadecanoic acid (C17:0) over 24 weeks. Serum FGF21 was quantified by ELISA with an observed range of 129–1599 pg/ml, but did not significantly change over the 24-week study period. Total adiponectin levels (mean ± SD) significantly increased from 776 ± 400 pmol/ml at week 0 to 1196 ± 467 pmol/ml at week 24. The percent unmodified adiponectin levels (mean ± SD) decreased from 23.8 ± 6.0% at week 0 to 15.2 ± 5.2% at week 24. Interestingly, although FGF21 levels did not change, there was a good correlation between FGF21 and total adiponectin (ρ = 0.788, P < 0.001). We quantified the abundances of serum ceramides and sphingosines (SPH) because adiponectin has a defined role in sphingolipid metabolism through adiponectin receptor-mediated activation of ceramidases. The most abundant ceramide in dolphin sera was Cer 24:1 comprising 49% of the ceramides measured. Significant reductions were observed in the unsaturated Cer 18:1, Cer 20:1, and Cer 24:1, whereas significant increases were observed in saturated Cer 22:0, Cer 24:0, and Cer 26:0. However, total serum ceramides did not change. Significant elevations were detected for total sphingosine, dihydrosphingosine, sphingosine-1-phosphate, and dihydrosphingosine-1-phosphate. Proteomic analysis of the serum proteins revealed few changes in serum proteins over the study period. In conclusion

  2. Co-option of the sphingolipid metabolism for the production of nitroalkene defensive chemicals in termite soldiers.

    PubMed

    Jirošová, Anna; Jančařík, Andrej; Menezes, Riya C; Bazalová, Olga; Dolejšová, Klára; Vogel, Heiko; Jedlička, Pavel; Buček, Aleš; Brabcová, Jana; Majer, Pavel; Hanus, Robert; Svatoš, Aleš

    2017-03-01

    The aliphatic nitroalkene (E)-1-nitropentadec-1-ene (NPD), reported in early seventies in soldiers of the termite genus Prorhinotermes, was the first documented nitro compound produced by insects. Yet, its biosynthetic origin has long remained unknown. Here, we investigated in detail the biosynthesis of NPD in P. simplex soldiers. First, we track the dynamics in major metabolic pathways during soldier ontogeny, with emphasis on likely NPD precursors and intermediates. Second, we propose a hypothesis of NPD formation and verify its individual steps using in vivo incubations of putative precursors and intermediates. Third, we use a de novo assembled RNA-Seq profiles of workers and soldiers to identify putative enzymes underlying NPD formation. And fourth, we describe the caste- and age-specific expression dynamics of candidate initial genes of the proposed biosynthetic pathway. Our observations provide a strong support to the following biosynthetic scenario of NPD formation, representing an analogy of the sphingolipid pathway starting with the condensation of tetradecanoic acid with l-serine and leading to the formation of a C16 sphinganine. The C16 sphinganine is then oxidized at the terminal carbon to give rise to 2-amino-3-hydroxyhexadecanoic acid, further oxidized to 2-amino-3-oxohexadecanoic acid. Subsequent decarboxylation yields 1-aminopentadecan-2-one, which then proceeds through six-electron oxidation of the amino moiety to give rise to 1-nitropentadecan-2-one. Keto group reduction and hydroxyl moiety elimination lead to NPD. The proposed biosynthetic sequence has been constructed from age-related quantitative dynamics of individual intermediates and confirmed by the detection of labeled products downstream of the administered labeled intermediates. Comparative RNA-Seq analyses followed by qRT-PCR validation identified orthologs of serine palmitoyltransferase and 3-ketodihydrosphingosine reductase genes as highly expressed in the NPD production site, i

  3. Altering sphingolipid composition with aging induces contractile dysfunction of gastric smooth muscle via K(Ca) 1.1 upregulation.

    PubMed

    Choi, Shinkyu; Kim, Ji Aee; Kim, Tae Hun; Li, Hai-Yan; Shin, Kyong-Oh; Lee, Yong-Moon; Oh, Seikwan; Pewzner-Jung, Yael; Futerman, Anthony H; Suh, Suk Hyo

    2015-12-01

    K(Ca) 1.1 regulates smooth muscle contractility by modulating membrane potential, and age-associated changes in K(Ca) 1.1 expression may contribute to the development of motility disorders of the gastrointestinal tract. Sphingolipids (SLs) are important structural components of cellular membranes whose altered composition may affect K(Ca) 1.1 expression. Thus, in this study, we examined whether altered SL composition due to aging may affect the contractility of gastric smooth muscle (GSM). We studied changes in ceramide synthases (CerS) and SL levels in the GSM of mice of varying ages and compared them with those in young CerS2-null mice. The levels of C16- and C18-ceramides, sphinganine, sphingosine, and sphingosine 1-phosphate were increased, and levels of C22, C24:1 and C24 ceramides were decreased in the GSM of both aged wild-type and young CerS2-null mice. The altered SL composition upregulated K(Ca) 1.1 and increased K(Ca) 1.1 currents, while no change was observed in K(Ca) 1.1 channel activity. The upregulation of KC a 1.1 impaired intracellular Ca²⁺mobilization and decreased phosphorylated myosin light chain levels, causing GSM contractile dysfunction. Additionally, phosphoinositide 3-kinase, protein kinase Cζ , c-Jun N-terminal kinases, and nuclear factor kappa-B were found to be involved in K(Ca) 1.1 upregulation. Our findings suggest that age-associated changes in SL composition or CerS2 ablation upregulate K(Ca) 1.1 via the phosphoinositide 3-kinase/protein kinase Cζ /c-Jun N-terminal kinases/nuclear factor kappa-B-mediated pathway and impair Ca²⁺ mobilization, which thereby induces the contractile dysfunction of GSM. CerS2-null mice exhibited similar effects to aged wild-type mice; therefore, CerS2-null mouse models may be utilized for investigating the pathogenesis of aging-associated motility disorders.

  4. Reproductive and sphingolipid metabolic effects of fumonisin B1 and its alkaline hydrolysis product in LM/Bc mice: hydrolyzed fumonisin B1 did not cause neural tube defects

    USDA-ARS?s Scientific Manuscript database

    Fumonisins are mycotoxins produced by Fusarium verticillioides. They are toxic to animals and exert their effects through mechanisms involving disruption of sphingolipid metabolism. Fumonisins and their hydrolyzed analogues are found in alkaline-cooked, maize-based foods such as tortillas and the c...

  5. A non-glycosylated and functionally deficient mutant (N215H) of the sphingolipid activator protein B (SAP-B) in a novel case of metachromatic leukodystrophy (MLD).

    PubMed

    Wrobe, D; Henseler, M; Huettler, S; Pascual Pascual, S I; Chabas, A; Sandhoff, K

    2000-02-01

    The lysosomal degradation of sphingolipids with short oligosaccharide chains depends on small glycosylated non-enzymatic sphingolipid activator proteins (SAPs, saposins). Four of the five known SAPs, SAP-A, -B, -C and -D, are derived by proteolytic processing from a common precursor protein (SAP-precursor) that is encoded by a gene on chromosome 10 consisting of 15 exons and 14 introns. SAP-B is a non-specific glycolipid binding protein that stimulates in vitro the hydrolysis of about 20 glycolipids by different enzymes. In vivo SAP-B stimulates in particular the degradation of sulphatides by arylsulphatase A. So far, four different point mutations have been identified on the SAP-B domain of the SAP-precursor gene. The mutations result in a loss of mature SAP-B, causing the lysosomal accumulation of sulphatides and other sphingolipids, resulting in variant forms of metachromatic leukodystrophy (MLD). Here we report on a patient with SAP-B deficiency that is caused by a new homoallelic point mutation that has been identified by mRNA and DNA analysis. A 643A > C transversion results in the exchange of asparagine 215 to histidine and eliminates the single glycosylation site of SAP-B. Metabolic labelling experiments showed that the mutation had no effect on the intracellular transport of the encoded precursor to the acidic compartments and its maturation in the patient's cells. All four SAPs (SAP-A to SAP-D) were detectable by immunochemical methods. SAP-B in the patient's cells was found to be slightly less stable than the protein in normal cells and corresponded in size to the deglycosylated form of the wild-type SAP-B. Feeding studies with non-glycosylated SAP-precursor, generating non-glycosylated SAP-B, showed that the loss of the carbohydrate chain reduced the intracellular activity of the protein significantly. The additional structural change of the patient's SAP-B, caused by the change of amino acid 215 from asparagine to histidine, presumably resulted in an

  6. Inhibition of sphingolipid synthesis improves dyslipidemia in the diet-induced hamster model of insulin resistance: evidence for the role of sphingosine and sphinganine in hepatic VLDL-apoB100 overproduction.

    PubMed

    Dekker, Mark J; Baker, Chris; Naples, Mark; Samsoondar, Josh; Zhang, Rianna; Qiu, Wei; Sacco, Jennifer; Adeli, Khosrow

    2013-05-01

    Sphingolipids have emerged as important bioactive lipid species involved in the pathogenesis of type 2 diabetes and cardiovascular disease. However, little is known of the regulatory role of sphingolipids in dyslipidemia of insulin-resistant states. We employed hamster models of dyslipidemia and insulin resistance to investigate the role of sphingolipids in hepatic VLDL overproduction, induction of insulin resistance, and inflammation. Hamsters were fed either a control chow diet, a high fructose diet, or a diet high in fat, fructose and cholesterol (FFC diet). They were then treated for 2 weeks with vehicle or 0.3 mg/kg myriocin, a potent inhibitor of de novo sphingolipid synthesis. Both fructose and FFC feeding induced significant increases in hepatic sphinganine, which was normalized to chow-fed levels with myriocin (P < 0.05); myriocin also lowered hepatic ceramide content (P < 0.05). Plasma TG and cholesterol as well as VLDL-TG and -apoB100 were similarly reduced with myriocin treatment in all hamsters, regardless of diet. Myriocin treatment also led to improved insulin sensitivity and reduced hepatic SREBP-1c mRNA, though it did not appear to ameliorate the activation of hepatic inflammatory pathways. Importantly, direct treatment of primary hamster hepatocytes ex vivo with C2 ceramide or sphingosine led to an increased secretion of newly synthesized apoB100. Taken together, these data suggest that a) hepatic VLDL-apoB100 overproduction may be stimulated by ceramides and sphingosine and b) inhibition of sphingolipid synthesis can reduce circulating VLDL in hamsters and improve circulating lipids--an effect that is possibly due to improved insulin signaling and reduced lipogenesis but is independent of changes in inflammation.

  7. A Lipidomics Approach to Assess the Association between Plasma Sphingolipids and Verbal Memory Performance in Coronary Artery Disease Patients Undertaking Cardiac Rehabilitation: A C18:0 Signature for Cognitive Response to Exercise.

    PubMed

    Saleem, Mahwesh; Herrmann, Nathan; Dinoff, Adam; Mielke, Michelle M; Oh, Paul I; Shammi, Prathiba; Cao, Xingshan; Venkata, Swarajya Lakshmi Vattem; Haughey, Norman J; Lanctôt, Krista L

    2017-06-03

    Early subtle deficits in verbal memory, which may indicate early neural risk, are common in patients with coronary artery disease (CAD). While exercise can improve cognition, cognitive response to exercise is heterogeneous. Sphingolipids have been associated with the development and progression of CAD, and impairments in sphingolipid metabolism may play roles in neurodegeneration and in the neural adaptation response to exercise. In this study, change in plasma concentrations of sphingolipids was assessed in relation to change in verbal memory performance and in other cognitive domains among CAD subjects undertaking a 6-month cardiac rehabilitation (CR) program. Patients with CAD (n = 120, mean age = 64±6 y, 84% male, years of education = 16±3) underwent CR with neuropsychological assessments and blood collected at baseline, 3-, and 6-months. Z-scores based on age, gender, and education were combined for verbal memory, visuospatial memory, processing speed, executive function, and global cognition tasks to calculate cognitive domain Z-scores. Plasma sphingolipid concentrations were measured from fasting blood samples using high performance liquid chromatography coupled electrospray ionization tandem mass spectrometry (LC/MS/MS). Mixed models were used to identify sphingolipids significantly associated with performance in verbal memory and other cognitive domains, adjusting for potential confounders. Lower ceramide C18:0 concentrations were significantly associated with improvement in verbal memory performance (b[SE] = -0.51 [0.25], p = 0.04), visuospatial memory (b[SE] = -0.44 [0.22], p = 0.05), processing speed (b[SE] = -0.89 [0.32], p = 0.007), and global cognition (b[SE] = -1.47 [0.59], p = 0.01) over 6 months of CR. Plasma ceramide C18:0 concentrations may be a sensitive marker of cognitive response to exercise in patients with CAD.

  8. TSC (Temperature Sensitive suppressors of the Calcium sensitivity of csg2 delta) Mutants; A Tool to Investigate Sphingolipid Metabolism in the Yeast Saccharomyces cerevisiae

    DTIC Science & Technology

    1998-08-14

    ofceralJ’ides within the cea or may alter the physical properties ofmembranes..19 They may also impact sphiDgotipid interaction with integral membrane...CJYaJO FAS2 TSC6 CJYaJ2 SCS7 TSC7 LHYall lJ a28, alOS ;LFrra34 SUR2 TSC8 Lf.ria33,837;LHYa33,alll,aI16lJ aI57,al66 SIN3 TSC9 LHYa39; LHYa38, a43, a6l lJ alOl...R.L., Dickson, R.C. (1997) Sphingolipid Synthesis as a Target for Antifungal Drugs. J. BioI. Chem. 272, 9809-9817 48. Hashida,...Okado, T., Ogawa, A

  9. Modification of sphingolipid metabolism by tamoxifen and N-desmethyltamoxifen in acute myelogenous leukemia – Impact on enzyme activity and response to cytotoxics

    PubMed Central

    Morad, Samy A. F.; Tan, Su-Fern; Feith, David J.; Kester, Mark; Claxton, David F.; Loughran, Thomas P.; Barth, Brian M.; Fox, Todd E.; Cabot, Myles C.

    2015-01-01

    The triphenylethylene antiestrogen, tamoxifen, can be an effective inhibitor of sphingolipid metabolism. This off-target activity makes tamoxifen an interesting ancillary for boosting the apoptosis-inducing properties of ceramide, a sphingolipid with valuable tumor censoring activity. Here we show for the first time that tamoxifen and metabolite, N –desmethyltamoxifen (DMT) block ceramide glycosylation and inhibit ceramide hydrolysis (by acid ceramidase, AC) in human acute myelogenous leukemia (AML) cell lines and in AML cells derived from patients. Tamoxifen (1-10 μM) inhibition of AC in AML cells was accompanied by decreases in AC protein expression. Tamoxifen also depressed expression and activity of sphingosine kinase 1 (SphK1), the enzyme catalyzing production of mitogenic sphingosine 1-phosphate (S1-P). Results from mass spectroscopy showed that tamoxifen and DMT, i ) increased the levels of endogenous C16:0- and C24:1 ceramide molecular species, ii) nearly totally halted production of respective glucosylceramide (GC) molecular species, iii ) drastically reduced levels of sphingosine ( to 9% of control), and iv ) reduced levels of S1-P by 85%, in vincristine-resistant HL-60/VCR cells. Co-administration of tamoxifen with either N-(4-hydroxyphenyl)retinamide (4-HPR), a ceramide-generating retinoid, or a cell-deliverable form of ceramide, C6-ceramide, resulted in marked decreases in HL-60/VCR cell viability that far exceeded single agent potency. Combination treatments resulted in synergistic apoptotic cell death as gauged by increased Annexin V binding and DNA fragmentation and activation of caspase-3. These results show the versatility of adjuvant triphenylethylene with ceramide-centric therapies for magnifying therapeutic potential in AML. Such drug regimens could serve as effective strategies, even in the multidrug resistant setting. PMID:25769964

  10. Modification of sphingolipid metabolism by tamoxifen and N-desmethyltamoxifen in acute myelogenous leukemia--Impact on enzyme activity and response to cytotoxics.

    PubMed

    Morad, Samy A F; Tan, Su-Fern; Feith, David J; Kester, Mark; Claxton, David F; Loughran, Thomas P; Barth, Brian M; Fox, Todd E; Cabot, Myles C

    2015-07-01

    The triphenylethylene antiestrogen, tamoxifen, can be an effective inhibitor of sphingolipid metabolism. This off-target activity makes tamoxifen an interesting ancillary for boosting the apoptosis-inducing properties of ceramide, a sphingolipid with valuable tumor censoring activity. Here we show for the first time that tamoxifen and metabolite, N-desmethyltamoxifen (DMT), block ceramide glycosylation and inhibit ceramide hydrolysis (by acid ceramidase, AC) in human acute myelogenous leukemia (AML) cell lines and in AML cells derived from patients. Tamoxifen (1-10 μM) inhibition of AC in AML cells was accompanied by decreases in AC protein expression. Tamoxifen also depressed expression and activity of sphingosine kinase 1 (SphK1), the enzyme-catalyzing production of mitogenic sphingosine 1-phosphate (S1-P). Results from mass spectroscopy showed that tamoxifen and DMT (i) increased the levels of endogenous C16:0 and C24:1 ceramide molecular species, (ii) nearly totally halted production of respective glucosylceramide (GC) molecular species, (iii) drastically reduced levels of sphingosine (to 9% of control), and (iv) reduced levels of S1-P by 85%, in vincristine-resistant HL-60/VCR cells. The co-administration of tamoxifen with either N-(4-hydroxyphenyl)retinamide (4-HPR), a ceramide-generating retinoid, or a cell-deliverable form of ceramide, C6-ceramide, resulted in marked decreases in HL-60/VCR cell viability that far exceeded single agent potency. Combination treatments resulted in synergistic apoptotic cell death as gauged by increased Annexin V binding and DNA fragmentation and activation of caspase-3. These results show the versatility of adjuvant triphenylethylene with ceramide-centric therapies for magnifying therapeutic potential in AML. Such drug regimens could serve as effective strategies, even in the multidrug-resistant setting.

  11. PDMP Blocks Brefeldin A–induced Retrograde Membrane Transport from Golgi to ER: Evidence for Involvement of Calcium Homeostasis and Dissociation from Sphingolipid Metabolism

    PubMed Central

    Kok, Jan Willem; Babia, Teresa; Filipeanu, Catalin M.; Nelemans, Adriaan; Egea, Gustavo; Hoekstra, Dick

    1998-01-01

    In this study, we show that an inhibitor of sphingolipid biosynthesis, d,l-threo-1-phenyl-2- decanoylamino-3-morpholino-1-propanol (PDMP), inhibits brefeldin A (BFA)-induced retrograde membrane transport from Golgi to endoplasmic reticulum (ER). If BFA treatment was combined with or preceded by PDMP administration to cells, disappearance of discrete Golgi structures did not occur. However, when BFA was allowed to exert its effect before PDMP addition, PDMP could not “rescue” the Golgi compartment. Evidence is presented showing that this action of PDMP is indirect, which means that the direct target is not sphingolipid metabolism at the Golgi apparatus. A fluorescent analogue of PDMP, 6-(N-[7-nitro-2,1,3-benzoxadiazol-4-yl]amino)hexanoyl-PDMP (C6-NBD-PDMP), did not localize in the Golgi apparatus. Moreover, the effect of PDMP on membrane flow did not correlate with impaired C6-NBD-sphingomyelin biosynthesis and was not mimicked by exogenous C6-ceramide addition or counteracted by exogenous C6-glucosylceramide addition. On the other hand, the PDMP effect was mimicked by the multidrug resistance protein inhibitor MK571. The effect of PDMP on membrane transport correlated with modulation of calcium homeostasis, which occurred in a similar concentration range. PDMP released calcium from at least two independent calcium stores and blocked calcium influx induced by either extracellular ATP or thapsigargin. Thus, the biological effects of PDMP revealed a relation between three important physiological processes of multidrug resistance, calcium homeostasis, and membrane flow in the ER/ Golgi system. PMID:9660860

  12. Impact of insulin deprivation and treatment on sphingolipid distribution in different muscle subcellular compartments of streptozotocin-diabetic C57Bl/6 mice

    PubMed Central

    Zabielski, Piotr; Blachnio-Zabielska, Agnieszka; Lanza, Ian R.; Gopala, Srinivas; Manjunatha, S.; Jakaitis, Daniel R.; Persson, Xuan-Mai; Gransee, Jaime; Klaus, Katherine A.; Schimke, Jill M.; Jensen, Michael D.

    2013-01-01

    Insulin deprivation in type 1 diabetes (T1D) individuals increases lipolysis and plasma free fatty acids (FFA) concentration, which can stimulate synthesis of intramyocellular bioactive lipids such as ceramides (Cer) and long-chain fatty acid-CoAs (LCFa-CoAs). Ceramide was shown to decrease muscle insulin sensitivity, and at mitochondrial levels it stimulates reactive oxygen species production. Here, we show that insulin deprivation in streptozotocin diabetic C57BL/6 mice increases quadriceps muscle Cer content, which was correlated with a concomitant decrease in the body fat and increased plasma FFA, glycosylated hemoglobin level (%Hb A1c), and muscular LCFa-CoA content. The alternations were accompanied by an increase in protein expression in LCFa-CoA and Cer synthesis (FATP1/ACSVL5, CerS1, CerS5), a decrease in the expression of genes implicated in muscle insulin sensitivity (GLUT4, GYS1), and inhibition of insulin signaling cascade by Aktα and GYS3β phosphorylation under acute insulin stimulation. Both the content and composition of sarcoplasmic fraction sphingolipids were most affected by insulin deprivation, whereas mitochondrial fraction sphingolipids remained stable. The observed effects of insulin deprivation were reversed, except for content and composition of LCFa-CoA, CerS protein expression, GYS1 gene expression, and phosphorylation status of Akt and GYS3β when exogenous insulin was provided by subcutaneous insulin implants. Principal component analysis and Pearson's correlation analysis revealed close relationships between the features of the diabetic phenotype, the content of LCFa-CoAs and Cers containing C18-fatty acids in sarcoplasm, but not in mitochondria. Insulin replacement did not completely rescue the phenotype, especially regarding the content of LCFa-CoA, or proteins implicated in Cer synthesis and muscle insulin sensitivity. These persistent changes might contribute to muscle insulin resistance observed in T1D individuals. PMID:24368672

  13. Inhibition of GSK3β by pharmacological modulation of sphingolipid metabolism occurs independently of ganglioside disturbance in a cellular model of Alzheimer's disease.

    PubMed

    Noel, Anastasia; Ingrand, Sabrina; Barrier, Laurence

    2015-09-01

    Accumulating evidence implicates ganglioside and/or related-sphingolipid disturbance in the pathogenesis of Alzheimer's disease (AD). However, it is not known whether these lipidic alterations are connected with other important features of AD, such as deregulated insulin/Akt/GSK3 signaling. In this study, we have treated neuroglioma cells expressing the double Swedish mutation of human amyloid precursor protein (H4APPsw) with several glycosphingolipid (GSL)-modulating agents, and we have analyzed the impact of the aberrant ganglioside composition on the GSK3 activation state. We found that both ceramide analogs D- and L-PDMP (1-phenyl 2-decanoylamino-3-morpholino-1-propanol), which have opposite effects on ganglioside synthesis, selectively inhibited GSK3β via Ser9 phosphorylation independently of the upstream insulin/Akt pathway. Conversely, the iminosugar N-butyldeoxynojirimycin (NB-DNJ) which displayed similar reduction of gangliosides as D-PDMP, did not affect the phosphorylation state of GSK3β. Concurrently, while NB-DNJ did not modify the cellular ceramide content, both PDMP enantiomers strongly and equally reduced the levels of long-chain ceramide species. Altogether, our findings led us to hypothesize that the PDMP-induced altered ganglioside composition is not the principal mechanism involved in the inhibition of GSK3β, but seems to implicate, at least in part, their ability to reduce ceramide levels. Nevertheless, this study provides new information regarding the possibilities to target GSK3β through modulation of sphingolipid metabolism. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Fumonisins disrupt sphingolipid metabolism, folate transport, and neural tube development in embryo culture and in vivo: a potential risk factor for human neural tube defects among populations consuming fumonisin-contaminated maize.

    PubMed

    Marasas, Walter F O; Riley, Ronald T; Hendricks, Katherine A; Stevens, Victoria L; Sadler, Thomas W; Gelineau-van Waes, Janee; Missmer, Stacey A; Cabrera, Julio; Torres, Olga; Gelderblom, Wentzel C A; Allegood, Jeremy; Martínez, Carolina; Maddox, Joyce; Miller, J David; Starr, Lois; Sullards, M Cameron; Roman, Ana Victoria; Voss, Kenneth A; Wang, Elaine; Merrill, Alfred H

    2004-04-01

    Fumonisins are a family of toxic and carcinogenic mycotoxins produced by Fusarium verticillioides (formerly Fusarium moniliforme), a common fungal contaminant of maize. Fumonisins inhibit ceramide synthase, causing accumulation of bioactive intermediates of sphingolipid metabolism (sphinganine and other sphingoid bases and derivatives) as well as depletion of complex sphingolipids, which interferes with the function of some membrane proteins, including the folate-binding protein (human folate receptor alpha). Fumonisin causes neural tube and craniofacial defects in mouse embryos in culture. Many of these effects are prevented by supplemental folic acid. Recent studies in LMBc mice found that fumonisin exposure in utero increases the frequency of developmental defects and administration of folate or a complex sphingolipid is preventive. High incidences of neural tube defects (NTD) occur in some regions of the world where substantial consumption of fumonisins has been documented or plausibly suggested (Guatemala, South Africa, and China); furthermore, a recent study of NTD in border counties of Texas found a significant association between NTD and consumption of tortillas during the first trimester. Hence, we propose that fumonisins are potential risk factors for NTD, craniofacial anomalies, and other birth defects arising from neural crest cells because of their apparent interference with folate utilization.

  15. Ca2+, Mg2+-dependent DNase involvement in apoptotic effects in spermatozoa of sea urchin Strongylocentrotus intermedius induced by two-headed sphingolipid rhizochalin.

    PubMed

    Sibirtsev, Juriy T; Shastina, Valeria V; Menzorova, Natalia I; Makarieva, Tatyana N; Rasskazov, Valeriy A

    2011-06-01

    Previously, we have purified three distinct DNases from spermatozoa of sea urchin Strongylocentrotus intermedius and we suppose the role of Ca(2+), Mg(2+)-dependent DNase (Ca, Mg-DNase) in apoptosis of spermatozoa. Two-headed sphingolipid rhizochalin (Rhz) induced characteristic apoptotic nuclear chromatin changes, internucleosomal DNA cleavage, and activation of caspase-9, caspase-8, and caspase-3 in spermatozoa as was shown by fluorescence Hoechst 33342/PI/FDA analysis, DNA fragmentation assay, and fluorescence caspase inhibitors FAM-LEHD-fmk, FAM-IETD-fmk, and FAM-DEVD-fmk, respectively. Inhibitor of caspase-3 z-DEVD-fmk subdued Rhz-induced internucleosomal ladder formation, which confirmed the major role of caspase-3 in apoptotic DNA cleavage probably through Ca, Mg-DNase activation. Participation of sea urchin Ca, Mg-DNase in apoptosis of spermatozoa was demonstrated by ions Zn(2+) blocking of Rhz-induced DNA fragmentation due to direct inhibition of the Ca, Mg-DNase and internucleosomal cleavage of HeLa S and Vero E6 cell nuclei chromatin by highly purified Ca, Mg-DNase.

  16. The ceramide activated protein phosphatase Sit4 impairs sphingolipid dynamics, mitochondrial function and lifespan in a yeast model of Niemann-Pick type C1.

    PubMed

    Vilaça, Rita; Barros, Ivo; Matmati, Nabil; Silva, Elísio; Martins, Telma; Teixeira, Vítor; Hannun, Yusuf A; Costa, Vítor

    2017-10-06

    The Niemann-Pick type C is a rare neurodegenerative disease that results from loss-of-function point mutations in NPC1 or NPC2, which affect the homeostasis of sphingolipids and sterols in human cells. We have previously shown that yeast lacking Ncr1, the orthologue of human NPC1 protein, display a premature ageing phenotype and higher sensitivity to oxidative stress associated with mitochondrial dysfunctions and accumulation of long chain bases. In this study, a lipidomic analysis revealed specific changes in the levels of ceramide species in ncr1Δ cells, including decreases in dihydroceramides and increases in phytoceramides. Moreover, the activation of Sit4, a ceramide-activated protein phosphatase, increased in ncr1Δ cells. Deletion of SIT4 or CDC55, its regulatory subunit, increased the chronological lifespan and hydrogen peroxide resistance of ncr1Δ cells and suppressed its mitochondrial defects. Notably, Sch9 and Pkh1-mediated phosphorylation of Sch9 decreased significantly in ncr1Δsit4Δ cells. These results suggest that phytoceramide accumulation and Sit4-dependent signaling mediate the mitochondrial dysfunction and shortened lifespan in the yeast model of Niemann-Pick type C1, in part through modulation of the Pkh1-Sch9 pathway. Copyright © 2017. Published by Elsevier B.V.

  17. FTY720 Induces Apoptosis of M2 Subtype Acute Myeloid Leukemia Cells by Targeting Sphingolipid Metabolism and Increasing Endogenous Ceramide Levels

    PubMed Central

    Li, Lianchun; Liu, Yuan-Fang; Wang, Jiang; Liu, Hong; Song, Heng; Jiang, Hualiang; Chen, Sai-Juan; Luo, Cheng; Li, Keqin Kathy

    2014-01-01

    The M2 subtype Acute Myeloid Leukemia (AML-M2) with t(8;21) represents an unmet challenge because of poor clinical outcomes in a sizable portion of patients. In this study,we report that FTY720 (Fingolimod), a sphingosine analogue and an FDA approved drug for treating of multiple sclerosis, shows antitumorigenic activity against the Kasumi-1 cell line, xenograft mouse models and leukemic blasts isolated from AML-M2 patients with t(8;21) translocation. Primary investigation indicated that FTY720 caused cell apoptosis through caspases and protein phosphatase 2A (PP2A) activation. Transcriptomic profiling further revealed that FTY720 treatment could upregulate AML1 target genes and interfere with genes involved in ceramide synthesis. Treatment with FTY720 led to the elimination of AML1-ETO oncoprotein and caused cell cycle arrest. More importantly, FTY720 treatment resulted in rapid and significant increase of pro-apoptotic ceramide levels, determined by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry based lipidomic approaches. Structural simulation model had also indicated that the direct binding of ceramide to inhibitor 2 of PP2A (I2PP2A) could reactivate PP2A and cause cell death. This study demonstrates, for the first time, that accumulation of ceramide plays a central role in FTY720 induced cell death of AML-M2 with t(8;21). Targeting sphingolipid metabolism by using FTY720 may provide novel insight for the drug development of treatment for AML-M2 leukemia. PMID:25050888

  18. Untargeted metabolomic analysis of human serum samples associated with exposure levels of Persistent organic pollutants indicate important perturbations in Sphingolipids and Glycerophospholipids levels.

    PubMed

    Carrizo, Daniel; Chevallier, Olivier P; Woodside, Jayne V; Brennan, Sarah F; Cantwell, Marie M; Cuskelly, Geraldine; Elliott, Christopher T

    2017-02-01

    Persistent organic pollutants (POPs) are distributed globally and are associated with adverse health effects in humans. A study combining gas chromatography-mass spectrometry (GC-MS), high resolution mass spectrometry (UPLC-QTof-MS) and chemometrics for the analysis of adult human serum samples was undertaken. Levels of serum POPs found were in the low range of what has been reported in similar populations across Europe (median 33.84 p, p'-DDE, 3.02 HCB, 83.55 β-HCH, 246.62 PCBs ng/g lipids). Results indicated that compounds concentrations were significantly different between the two groups of POPs exposure (high vs low) and classes (DDE, β-HCH, HCB, PCBs). Using orthogonal partial last-squares discriminant analysis (OPLS-DA), multivariate models were created for both modes of acquisition and POPs classes, explaining the maximum amount of variation between sample groups (positive mode R2 = 98-90%; Q2 = 94-75%; root mean squared error of validation (RMSEV) = 12-20%: negative mode R2 = 98-91%; Q2 = 94-81%; root mean squared error of validation (RMSEV) = 10-19%. In the serum samples analyzed, a total 3076 and 3121 ions of interest were detected in positive and negative mode respectively. Of these, 40 were found to be significantly different (p < 0.05) between exposure levels. Sphingolipids and Glycerophospholipids lipids families were identified and found significantly (p < 0.05) different between high and low POPs exposure levels. This study has shown that the elucidation of metabolomic fingerprints may have the potential to be classified as biomarkers of POPs exposure. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Enigmol: A novel sphingolipid analog with anti-cancer activity against cancer cell lines and in vivo models for intestinal and prostate cancer

    PubMed Central

    Symolon, Holly; Bushnev, Anatoliy; Peng, Qiong; Ramaraju, Harsha; Mays, Suzanne G.; Allegood, Jeremy C.; Pruett, Sarah T.; Sullards, M. Cameron; Dillehay, Dirck L.; Liotta, Dennis C.; Merrill, Alfred H.

    2017-01-01

    Sphingoid bases are cytotoxic for many cancer cell lines, and are thought to contribute to suppression of intestinal tumorigenesis in vivo by ingested sphingolipids. This study explored the behavior of a sphingoid base analog, (2S,3S,5S)-2-amino-3,5-dihydroxyoctadecane (“Enigmol”), that cannot be phosphorylated by sphingosine kinases and is slowly N-acylated, therefore, is more persistent than natural sphingoid bases. Enigmol had potential anti-cancer activity in a National Cancer Institute (NCI-60) cell line screen, and was confirmed to be more cytotoxic and persistent than naturally occurring sphingoid bases using HT29 cells, a colon cancer cell line. Although the molecular targets of sphingoid bases are not well delineated, Enigmol shared one of the mechanisms that has been found for naturally occurring sphingoid bases: to “normalize” the aberrant accumulation of β-catenin in the nucleus and cytoplasm of colon cancer cells due to defect(s) in the adenomatous polyposis coli (APC)/β-catenin regulatory system. Enigmol also had anti-tumor efficacy when administered orally to Min mice, a mouse model with a truncated APC gene product (C57Bl/6JMin/+ mice), decreasing the number of intestinal tumors by half at 0.025 % of the diet (w/w), with no evidence of host toxicity until higher dosages. Enigmol was also tested against the prostate cancer cell lines DU145 and PC-3 in nude mouse xenografts, and suppressed tumor growth in both. Thus, Enigmol represents a novel category of sphingoid base analog that is orally bioavailable and has the potential to be effective against multiple types of cancer. PMID:21398423

  20. A comprehensive lipidomic screen of pancreatic β-cells using mass spectroscopy defines novel features of glucose-stimulated turnover of neutral lipids, sphingolipids and plasmalogens.

    PubMed

    Pearson, Gemma L; Mellett, Natalie; Chu, Kwan Yi; Boslem, Ebru; Meikle, Peter J; Biden, Trevor J

    2016-06-01

    Glucose promotes lipid remodelling in pancreatic β-cells, and this is thought to contribute to the regulation of insulin secretion, but the metabolic pathways and potential signalling intermediates have not been fully elaborated. Using mass spectrometry (MS) we quantified changes in approximately 300 lipid metabolites in MIN6 β-cells and isolated mouse islets following 1 h stimulation with glucose. Flux through sphingolipid pathways was also assessed in (3)H-sphinganine-labelled cells using TLC. Glucose specifically activates the conversion of triacylglycerol (TAG) to diacylglycerol (DAG). This leads indirectly to the formation of 18:1 monoacylglycerol (MAG), via degradation of saturated/monounsaturated DAG species, such as 16:0_18:1 DAG, which are the most abundant, immediate products of glucose-stimulated TAG hydrolysis. However, 16:0-containing, di-saturated DAG species are a better direct marker of TAG hydrolysis since, unlike the 18:1-containing DAGs, they are predominately formed via this route. Using multiple reaction monitoring, we confirmed that in islets under basal conditions, 18:1 MAG is the most abundant species. We further demonstrated a novel site of glucose to enhance the conversion of ceramide to sphingomyelin (SM) and galactosylceramide (GalCer). Flux and product:precursor analyses suggest regulation of the enzyme SM synthase, which would constitute a separate mechanism for localized generation of DAG in response to glucose. Phosphatidylcholine (PC) plasmalogen (P) species, specifically those containing 20:4, 22:5 and 22:6 side chains, were also diminished in the presence of glucose, whereas the more abundant phosphatidylethanolamine plasmalogens were unchanged. Our results highlight 18:1 MAG, GalCer, PC(P) and DAG/SM as potential contributors to metabolic stimulus-secretion coupling.

  1. FTY720 induces apoptosis of M2 subtype acute myeloid leukemia cells by targeting sphingolipid metabolism and increasing endogenous ceramide levels.

    PubMed

    Chen, Limin; Luo, Liu-Fei; Lu, Junyan; Li, Lianchun; Liu, Yuan-Fang; Wang, Jiang; Liu, Hong; Song, Heng; Jiang, Hualiang; Chen, Sai-Juan; Luo, Cheng; Li, Keqin Kathy

    2014-01-01

    The M2 subtype Acute Myeloid Leukemia (AML-M2) with t(8;21) represents an unmet challenge because of poor clinical outcomes in a sizable portion of patients. In this study,we report that FTY720 (Fingolimod), a sphingosine analogue and an FDA approved drug for treating of multiple sclerosis, shows antitumorigenic activity against the Kasumi-1 cell line, xenograft mouse models and leukemic blasts isolated from AML-M2 patients with t(8;21) translocation. Primary investigation indicated that FTY720 caused cell apoptosis through caspases and protein phosphatase 2A (PP2A) activation. Transcriptomic profiling further revealed that FTY720 treatment could upregulate AML1 target genes and interfere with genes involved in ceramide synthesis. Treatment with FTY720 led to the elimination of AML1-ETO oncoprotein and caused cell cycle arrest. More importantly, FTY720 treatment resulted in rapid and significant increase of pro-apoptotic ceramide levels, determined by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry based lipidomic approaches. Structural simulation model had also indicated that the direct binding of ceramide to inhibitor 2 of PP2A (I2PP2A) could reactivate PP2A and cause cell death. This study demonstrates, for the first time, that accumulation of ceramide plays a central role in FTY720 induced cell death of AML-M2 with t(8;21). Targeting sphingolipid metabolism by using FTY720 may provide novel insight for the drug development of treatment for AML-M2 leukemia.

  2. The role of the trans double bond in skin barrier sphingolipids: permeability and infrared spectroscopic study of model ceramide and dihydroceramide membranes.

    PubMed

    Skolová, Barbora; Jandovská, Kateřina; Pullmannová, Petra; Tesař, Ondřej; Roh, Jaroslav; Hrabálek, Alexandr; Vávrová, Kateřina

    2014-05-20

    Dihydroceramides (dCer) are members of the sphingolipid family that lack the C4 trans double bond in their sphingoid backbone. In addition to being precursors of ceramides (Cer) and phytoceramides, dCer have also been found in the extracellular lipid membranes of the epidermal barrier, the stratum corneum. However, their role in barrier homeostasis is not known. We studied how the lack of the trans double bond in dCer compared to Cer influences the permeability, lipid chain order, and packing of multilamellar membranes composed of the major skin barrier lipids: (d)Cer, fatty acids, cholesterol, and cholesteryl sulfate. The permeability of the membranes with long-chain dCer was measured using various markers and was either comparable to or only slightly greater than (by up to 35%, not significant) that of the Cer membranes. The dCer were less sensitive to acyl chain shortening than Cer (the short dCer membranes were up to 6-fold less permeable that the corresponding short Cer membranes). Infrared spectroscopy showed that long dCer mixed less with fatty acids but formed more thermally stable ordered domains than Cer. The key parameter explaining the differences in permeability in the short dCer and Cer was the proportion of the orthorhombic phase. Our results suggest that the presence of the trans double bond in Cer is not crucial for the permeability of skin lipid membranes and that dCer may be underappreciated members of the stratum corneum lipid barrier that increase its heterogeneity.

  3. Enhanced killing of SCC17B human head and neck squamous cell carcinoma cells after photodynamic therapy plus fenretinide via the de novo sphingolipid biosynthesis pathway and apoptosis.

    PubMed

    Boppana, Nithin B; Stochaj, Ursula; Kodiha, Mohamed; Bielawska, Alicja; Bielawski, Jacek; Pierce, Jason S; Korbelik, Mladen; Separovic, Duska

    2015-05-01

    Because photodynamic therapy (PDT) alone is not always effective as an anticancer treatment, PDT is combined with other anticancer agents for improved efficacy. The clinically-relevant fenretinide [N-(4-hydroxyphenyl) retinamide; 4HPR], was combined with the silicon phthalocyanine photosensitizer Pc4-mediated PDT to test for their potential to enhance killing of SCC17B cells, a clinically-relevant model of human head and neck squamous cell carcinoma. Because each of these treatments induces apoptosis and regulates the de novo sphingolipid (SL) biosynthesis pathway, the role of ceramide synthase, the pathway-associated enzyme, in PDT+4HPR-induced apoptotic cell death was determined using the ceramide synthase inhibitor fumonisin B1 (FB). PDT+4HPR enhanced loss of clonogenicity. zVAD-fmk, a pan-caspase inhibitor, and FB, protected cells from death post-PDT+4HPR. In contrast, the anti-apoptotic protein Bcl2 inhibitor ABT199 enhanced cell killing after PDT+4HPR. Combining PDT with 4HPR led to FB-sensitive, enhanced Bax associated with mitochondria and cytochrome c redistribution. Mass spectrometry data showed that the accumulation of C16-dihydroceramide, a precursor of ceramide in the de novo SL biosynthesis pathway, was enhanced after PDT+4HPR. Using quantitative confocal microscopy, we found that PDT+4HPR enhanced dihydroceramide/ceramide accumulation in the ER, which was inhibited by FB. The results suggest that SCC17B cells are sensitized to PDT by 4HPR via the de novo SL biosynthesis pathway and apoptosis, and imply potential clinical relevance of the combination for cancer treatment.

  4. Enhanced killing of SCC17B human head and neck squamous cell carcinoma cells after photodynamic therapy plus fenretinide via the de novo sphingolipid biosynthesis pathway and apoptosis

    PubMed Central

    BOPPANA, NITHIN B.; STOCHAJ, URSULA; KODIHA, MOHAMED; BIELAWSKA, ALICJA; BIELAWSKI, JACEK; PIERCE, JASON S.; KORBELIK, MLADEN; SEPAROVIC, DUSKA

    2015-01-01

    Because photodynamic therapy (PDT) alone is not always effective as an anticancer treatment, PDT is combined with other anticancer agents for improved efficacy. The clinically-relevant fenretinide [N-(4-hydroxyphenyl) retinamide; 4HPR], was combined with the silicon phthalocyanine photosensitizer Pc4-mediated PDT to test for their potential to enhance killing of SCC17B cells, a clinically-relevant model of human head and neck squamous cell carcinoma. Because each of these treatments induces apoptosis and regulates the de novo sphingolipid (SL) biosynthesis pathway, the role of ceramide synthase, the pathway-associated enzyme, in PDT+4HPR-induced apoptotic cell death was determined using the ceramide synthase inhibitor fumonisin B1 (FB). PDT+4HPR enhanced loss of clonogenicity. zVAD-fmk, a pan-caspase inhibitor, and FB, protected cells from death post-PDT+4HPR. In contrast, the anti-apoptotic protein Bcl2 inhibitor ABT199 enhanced cell killing after PDT+4HPR. Combining PDT with 4HPR led to FB-sensitive, enhanced Bax associated with mitochondria and cytochrome c redistribution. Mass spectrometry data showed that the accumulation of C16-dihydroceramide, a precursor of ceramide in the de novo SL biosynthesis pathway, was enhanced after PDT+4HPR. Using quantitative confocal microscopy, we found that PDT+4HPR enhanced dihydroceramide/ceramide accumulation in the ER, which was inhibited by FB. The results suggest that SCC17B cells are sensitized to PDT by 4HPR via the de novo SL biosynthesis pathway and apoptosis, and imply potential clinical relevance of the combination for cancer treatment. PMID:25739041

  5. Non-dioxin-like organic toxicant PCB153 modulates sphingolipid metabolism in liver progenitor cells: its role in Cx43-formed gap junction impairment.

    PubMed

    Pierucci, F; Frati, A; Squecco, R; Lenci, E; Vicenti, C; Slavik, J; Francini, F; Machala, M; Meacci, E

    2017-02-01

    The non-dioxin-like environmental toxicant 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153), member of a group of persistent organic pollutants wide-spread throughout the environment, reduces gap junction intercellular communication (GJIC), an event possibly associated with tumor promotion. Since very few studies have investigated the signaling effectors and mode(s) of action of PCB153, and it is known that the gap junction (GJ) protein Cx43 can be regulated by the bioactive sphingolipid (SL) sphingosine 1-phosphate (S1P), this in vitro study mainly addresses whether SL metabolism is affected by PCB153 in rat liver epithelial WB-F344 cells. PCB153 treatment obtained significant changes in the S1P/ceramide (Cer) ratio, known to be crucial in determining cell fate. In particular, an increase in S1P at 30 min and a decrease of the bioactive lipid at 3 h were observed, whereas Cer level increased at 1 h and 24 h. Notably, a time-dependent modulation of sphingosine kinase (SphK), the enzyme responsible for S1P synthesis, and of its regulators, ERK1/2 and protein phosphatase PP2A, supports the involvement of these signaling effectors in PCB153 toxicity. Electrophysiological analyses, furthermore, indicated that the lipophilic environmental toxicant significantly reduced GJ biophysical properties, affecting both voltage-dependent (such as those formed by Cx43 and/or Cx32) and voltage-independent channels, thereby demonstrating that PCB153 may act differently on GJs formed by distinct Cx isoforms. SphK down-regulation alone induced GJIC impairment, and, when combined with PCB153, the acute effect on GJ suppression was additive. Moreover, after enzyme-specific gene silencing, the SphK1 isoform appears to be responsible for down-regulating Cx43 expression, while being the target of PCB153 at short-term exposure. In conclusion, we provide the first evidence of novel effectors in PCB153 toxic action in rat liver stem-like cells, leading us to consider SLs as potential markers

  6. Direct interaction between the inhibitor 2 and ceramide via sphingolipid-protein binding is involved in the regulation of protein phosphatase 2A activity and signaling

    PubMed Central

    Mukhopadhyay, Archana; Saddoughi, Sahar A.; Song, Pengfei; Sultan, Iyad; Ponnusamy, Suriyan; Senkal, Can E.; Snook, Christopher F.; Arnold, Hugh K.; Sears, Rosalie C.; Hannun, Yusuf A.; Ogretmen, Besim

    2009-01-01

    In this study, the inhibitor 2 of protein phosphatase 2A (I2PP2A) was identified in vitro and in situ as a ceramide-binding protein, which exhibits stereoisomer specificity and fatty acid chain length preference. Site- directed mutagenesis coupled with structural details of I2PP2A suggested that VIK 207-209 residues localized on helix 7 are important for ceramide binding and single mutation of K209D altered this interaction. Notably, I2PP2A-ceramide binding decreased the association between PP2A and the inhibitor, preventing the inhibition of PP2A activity in vitro. In addition, studies in A549 human lung cancer cells revealed that ceramide mediates c-Myc degradation via its PP2A-dependent dephosphorylation at S62, and treatment with okadaic acid and expression of c-Myc mutants with S62A or S62D conversions resulted in resistance to ceramide-mediated degradation. Importantly, whereas down-regulation of I2PP2A enhanced PP2A-mediated c-Myc degradation in response to ceramide, ectopic expression of wild-type I2PP2A but not of its K209D mutant protected this degradation in A549 cells. Moreover, expression of wild-type I2PP2A prevented the growth-inhibitory effects of ceramide both against A549 cells and xenograft-driven tumors in situ and in vivo compared with that in controls. Thus, these results suggest that direct interaction of I2PP2A with ceramide plays important biological roles via the regulation of PP2A activity and signaling, which in turn control ceramide-mediated degradation of c-Myc and antiproliferation.—Mukhopadhyay, A., Saddoughi, S. A., Song, P., Sultan, I., Ponnusamy, S., Senkal, C. E., Snook, C. F., Arnold, H. K., Sears, R. C., Hannun, Y. A., Ogretmen, B. Direct interaction between the inhibitor 2 and ceramide via sphingolipid-protein binding is involved in the regulation of protein phosphatase 2A activity and signaling. PMID:19028839

  7. Intracellular translocation of fluorescent sphingolipids in cultured fibroblasts: endogenously synthesized sphingomyelin and glucocerebroside analogues pass through the Golgi apparatus en route to the plasma membrane

    PubMed Central

    1985-01-01

    When monolayer cultures of Chinese hamster lung fibroblasts are briefly incubated at 2 degrees C with the fluorescent sphingolipid analogue, C6- NBD-ceramide (N- [7-(4-nitrobenzo-2-oxa-1,3-diazole)] aminocaproyl sphingosine), fluorescent labeling of the mitochondria, endoplasmic reticulum, and nuclear envelope occur. During further incubation at 37 degrees C, the Golgi apparatus, and later the plasma membrane, become intensely fluorescent. Within this period, the C6-NBD-ceramide is converted to equal amounts of fluorescent sphingomyelin and glucocerebroside (Lipsky, N. G., and R. E. Pagano, 1983, Proc. Natl. Acad. Sci. USA., 80:2608-2612). In the present study, the intracellular translocation of these metabolites and their subsequent appearance at the plasma membrane were investigated by fluorescence microscopy, the addition of the ionophore monensin, and the technique of "back exchange," in which the amounts and types of fluorescent lipids present at the cell surface are identified after their transfer from the cell surface into recipient vesicles. In control cells, the amount of fluorescent glucocerebroside and sphingomyelin that could be removed from the cell surface by back exchange increased during incubation at 37 degrees C, correlating with the increased fluorescence of the plasma membrane observed by microscopy. In the presence of 10 microM monensin, visible labeling of the plasma membrane was greatly diminished, whereas the Golgi apparatus became highly fluorescent and distended. The ability to remove fluorescent metabolites from the cell surface by back exchange was significantly but reversibly inhibited by monensin. Monensin also increased the total amount of fluorescent sphingomyelin, but not the glucocerebroside found in cells. Subcellular fractions were assayed for their ability to convert radiolabeled and fluorescent ceramides to the corresponding sphingomyelins and glucocerebrosides. The activities of parallel fractions coincided, suggesting that

  8. Characterization of sphingolipids from mycopathogens: factors correlating with expression of 2-hydroxy fatty acyl (E)-Delta 3-unsaturation in cerebrosides of Paracoccidioides brasiliensis and Aspergillus fumigatus.

    PubMed

    Toledo, M S; Levery, S B; Straus, A H; Suzuki, E; Momany, M; Glushka, J; Moulton, J M; Takahashi, H K

    1999-06-01

    results are discussed with respect to possible functional roles for fungal sphingolipids, particularly as they relate to the morphological transitions exhibited by P. brasiliensis.

  9. Direct and maternal n-3 long-chain polyunsaturated fatty acid supplementation improved triglyceridemia and glycemia through the regulation of hepatic and muscle sphingolipid synthesis in offspring hamsters fed a high-fat diet.

    PubMed

    Kasbi-Chadli, Fatima; Ferchaud-Roucher, Véronique; Krempf, Michel; Ouguerram, Khadija

    2016-03-01

    We recently reported that direct and maternal supplementation with n-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA) alleviates the metabolic disturbances in adult hamster pups fed with a high-fat diet (HFD). In this study, we hypothesized that these results involved a perinatal modulating effect of sphingolipids by n-3 LC-PUFA. We studied the effect of direct and maternal n-3 LC-PUFA supplementation on sphingolipid contents in liver and muscle, hepatic triglycerides (TG) secretion and glucose tolerance. Offspring male hamsters born from supplemented (Cω) or unsupplemented (C) mothers were subjected after weaning to a HFD during 16 weeks, without (Cω-HF or C-HF) or with direct supplementation with n-3 LC-PUFA (C-HFω). Direct supplementation decreased sphingosine, sphinganine and ceramides in liver and decreased sphingosine, sphinganine, sphingosine-1-phosphate (S1P) and ceramides in muscle in C-HFω compared to C-HF (p < 0.05). Maternal supplementation decreased C20 ceramide and lactosylceramide in liver and sphinganine, S1P and lactosylceramide in muscle (p < 0.05). This supplementation tended to decrease glucosylceramide in liver (p < 0.06) and muscle (p < 0.07) in Cω-HF compared to C-HF. Direct supplementation increased glucose tolerance and decreased hepatic TG secretion and hepatic gene expression levels of diacylglycerol O-acyltransferase 2 (DGAT2), sterol regulatory element-binding protein-1c (SREBP-1c), fatty acid synthase, stearoyl-CoA desaturase-1 (SCD1) and tumor necrosis factor α (TNFα). Maternal supplementation decreased basal glycemia and hepatic TG secretion. We observed a positive correlation between hepatic TG secretion and hepatic ceramide (p = 0.0059), and between basal glycemia and hepatic ceramide (p = 0.04) or muscle lactosylceramide contents (p = 0.001). We observed an improvement of lipids and glucose metabolism in hamster with n-3 LC-PUFA direct supplementation and a decrease in glycemia and hepatic TG secretion with maternal

  10. Short-term Mg deficiency upregulates protein kinase C isoforms in cardiovascular tissues and cells; relation to NF-kB, cytokines, ceramide salvage sphingolipid pathway and PKC-zeta: hypothesis and review.

    PubMed

    Altura, Burton M; Shah, Nilank C; Shah, Gatha J; Zhang, Aimin; Li, Wenyan; Zheng, Tao; Perez-Albela, Jose Luis; Altura, Bella T

    2014-01-01

    Numerous recent,epidemiological studies reveal that Western populations are growing more and more deficient in daily Mg intake which have been linked to etiology of cardiovascular (CV) diseases. A growing body of evidence suggests that a major missing link to this dilemma may reside within the sphingolipid-ceramide pathways. For the past 25 years , our labs have been focusing on these pathways in Mg-deficient mammals. The objective of this paper is two-fold: 1) to test various hypotheses and 2) to review the current status of the field and how protein kinase C isoforms may be pivotal to solving some of the CV attributes of Mg deficiency. Below, we test the hypotheses that: 1) short-term dietary deficiency of magnesium (MgD) would result in the upregulation of protein kinase C (PKC) isoforms in left ventricular (LV) and aortic smooth muscle (ASM) and serum; 2) MgD would result in a release of select cytokines and an upregulation of NF-kB in LV and ASM, and in primary cultured aortic smooth muscle cells (PCASMC); 3) MgD would result in an activation of the sphingolipid salvage pathway in LV and ASM, and in PCASMC; 4) MgD would result in a synthesis of sphingosine, but not sphinganine, in PCASMC which could be inhibited by fumonisin B1 (FB) an inhibitor of ceramide synthase (CS), but not scyphostatin an inhibitor of neutral sphingomyelinase (N-SMase); 5) incubation of PCASMC (in low Mg(2+)) with the PKC-mimic PMA would result in release and synthesis of NF-kB, cytokines, and ceramide but not sphingosine. The new data indicate that short-term MgD (10% normal dietary intake) result in an upregulation of all three classes of PKC isoforms in LV, aortic muscle and in serum coupled to the upregulation of ceramide, NF-kB activation, and cytokines. High degrees of linear correlation were found to exist between upregulation of PKC isoforms, p65 and cytokine release, suggesting cross-talk between these molecules and molecular pathways. Our experiments with PCASMCs demonstrated

  11. In-Depth Lipidomic Analysis of Molecular Species of Triacylglycerides, Diacylglycerides, Glycerophospholipids, and Sphingolipids of Buttermilk by GC-MS/FID, HPLC-ELSD, and UPLC-QToF-MS

    PubMed Central

    Castro-Gómez, Pilar; Montero, Olimpio; Fontecha, Javier

    2017-01-01

    Buttermilk, a byproduct of butter manufacturing, has gained considerable attention due to its high concentration of polar lipids as phospho- and sphingolipids from the milk fat globule membrane (MFGM). These polar lipids (PLs) are essential components of all cellular membranes and exert a variety of indispensable metabolic, neurological, and intracellular signaling processes. Despite its importance, there are few research studies that report a comprehensive characterization of the lipid molecular species of MFGM that could contribute to a better understanding of their putative healthful activities. In this study, procedures such as pressurized liquid extraction of polar and nonpolar lipids and their fractionation by flash chromatography have been carried out. The obtained fractions were submitted to an exhaustive characterization from a lipidomic point of view. The characterization includes new data about the identification and quantification of triacylglycerides (TAG), diacylglycerides (DAG), and phospho- and sphingolipids using different chromatographic techniques. The fatty acid profile was comparable to that of the milk fat but with a highly diverse composition of fatty acids. Molecular species have also been determined by using ultra-high performance liquid chromatography/quadruple-time-of-flight mass spectrometry (UPLC/QToF-MS). The TAG (16:0/16:0/6:0) and TAG (16:0/16:0/8:0) were the predominant saturated TAG species and TAG (14:0/18:1/16:0) and TAG (16:0/16:0/18:1) presented the highest content of monounsaturated TAG species. Furthermore; over 30 molecular species of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositol (PI) could be identified within PL, with PC (16:0/18:1) being the most abundant species. Whereas C16:0 was found to be the preferred FA in TAGs, it was C18:1 in PLs. Several ganglioside species have also been characterized with d18:1 ceramide moiety and secondary acyl chains ranging from C

  12. Sphingolipid metabolism in organotypic mouse keratinocyte cultures

    SciTech Connect

    Madison, K.C.; Swartzendruber, D.C.; Wertz, P.W.; Downing, D.T. )

    1990-12-01

    Ceramides are the dominant component of the stratum corneum intercellular lipid lamellae, which constitute the epidermal permeability barrier. Only pig and human epidermal ceramides have been extensively characterized and the structures of the ceramides of cultured keratinocytes have not been previously investigated. In the present studies, we have characterized the ceramides synthesized by organotypic lifted mouse keratinocyte cultures for the first time and compared them to the ceramides of intact mouse epidermis. Both mouse epidermis and cultures contained five ceramides, ceramide 1 being the least polar and ceramide 5 the most polar. Ceramide 1 was a group of acylceramides, i.e., very-long-chain omega-hydroxyceramides with an ester-linked nonhydroxy fatty acid. Ceramide 2 contained medium-length saturated nonhydroxy fatty acids. (In culture, the ceramide 2 band was split into two parts with the slightly more polar ceramide 2' containing short-chain saturated nonhydroxy fatty acids.) Ceramide 5 contained short-chain alpha-hydroxy fatty acids. The structures of ceramides 1, 2, and 5 were analagous to those of pig and human epidermis. Mouse epidermal ceramide 3 was quite unusual, containing beta-hydroxy fatty acids, a structure not previously identified among mammalian ceramides. In contrast, culture ceramide 3 was composed of omega-hydroxy fatty acids with a chain-length distribution similar to that of ceramide 1. Mouse ceramide 4 was composed of fatty acids with chromatographic mobility similar to hydroxy fatty acids but with different chemical reactivity; it remains only partially characterized. Culture ceramide 4 was present in quantities too small for analysis. All ceramides in mouse epidermis and cultures contained only sphingosine bases, whereas pig and human ceramides also contain phytosphingosine.

  13. Sphingolipid transfer proteins defined by the GLTP-fold

    PubMed Central

    Malinina, Lucy; Simanshu, Dhirendra K.; Zhai, Xiuhong; Samygina, Valeria R.; Kamlekar, RaviKanth; Kenoth, Roopa; Ochoa-Lizarralde, Borja; Malakhova, Margarita L.; Molotkovsky, Julian G.; Patel, Dinshaw J.; Brown, Rhoderick E.

    2015-01-01

    Glycolipid transfer proteins (GLTPs) originally were identified as small (~24 kDa), soluble, amphitropic proteins that specifically accelerate the intermembrane transfer of glycolipids. GLTPs and related homologs now are known to adopt a unique, helically dominated, two-layer ‘sandwich’ architecture defined as the GLTP-fold that provides the structural underpinning for the eukaryotic GLTP superfamily. Recent advances now provide exquisite insights into structural features responsible for lipid headgroup selectivity as well as the adaptability of the hydrophobic compartment for accommodating hydrocarbon chains of differing length and unsaturation. A new understanding of the structural versatility and evolutionary premium placed on the GLTP motif has emerged. Human GLTP-motifs have evolved to function not only as glucosylceramide binding/transferring domains for phosphoinositol 4-phosphate adaptor protein-2 during glycosphingolipid biosynthesis but also as selective binding/transfer proteins for ceramide-1-phosphate. The latter, known as ceramide-l-phosphate transfer protein, recently has been shown to form GLTP-fold while critically regulating Group-IV cytoplasmic phospholipase A2 activity and pro-inflammatory eicosanoid production. PMID:25797198

  14. Comparison of the Sphingolipid Content of Rumen Bacteroides Species

    PubMed Central

    Kunsman, Joseph E.; Caldwell, Daniel R.

    1974-01-01

    Ten strains of Bacteroides ruminicola were found to contain phosphosphingolipids. Four strains of Bacteroides amylophilus and one strain each of Bacteroides succinogenes and Bacteroides sp. were devoid of phosphosphingolipids. PMID:4476196

  15. Ischemia/Reperfusion Injury Alters Sphingolipid Metabolism in the Gut.

    PubMed

    Hoehn, Richard S; Seitz, Aaron P; Jernigan, Peter L; Gulbins, Erich; Edwards, Michael J

    2016-01-01

    Intestinal ischemia/reperfusion injury (I/R) is a significant cause of morbidity and mortality in surgical patients. Ceramide is a mediator of apoptosis and has been implicated as increasing bacterial infection susceptibility. The metabolite of ceramide, sphingosine, was recently shown to play an important role in the cell-autonomous, innate immune response of the upper respiratory tract by killing bacterial pathogens. The role of ceramide and/or sphingosine after mesenteric I/R is unknown. We investigated the specific effects of intestinal I/R on tissue ceramide and sphingosine concentration and resulting susceptibility to bacterial invasion. To simulate intestinal I/R, C57BL/6 mice underwent 30 minutes of vascular clamp-induced occlusion of the superior mesenteric artery followed by variable reperfusion times. Jejunum segments and intraluminal contents were analyzed for ceramide, sphingosine and bacteria using immunohistochemistry. Jejunum samples were also homogenized and cultured to quantify bacterial presence in the proximal intestine. We hypothesized that I/R induces an increase of ceramide in the intestine resulting in increased permeability, while a concomitant decrease of sphingosine may permit bacterial overgrowth. Control mice had no measurable bacteria in their proximal jejunum as measured by tissue culture and immunohistochemistry. After I/R, bacterial counts in the jejunum increased in a time-dependent manner, reaching a peak at 12 hours after reperfusion. Immunohistochemical analysis revealed a marked increase in ceramide in the vasculature of jejunal villi. In contrast, while ceramide concentrations in the epithelial cells decreased after I/R, sphingosine levels appeared to remain unchanged. Surprisingly, bacteria present in the jejunal lumen following I/R contained a ceramide coat. These data indicate that intestinal I/R leads to small intestine bacterial overgrowth as well as ceramide formation in the jejunal vasculature, which may contribute to the gut permeability associated with this injury. Moreover, our novel finding of ceramide in bacterial membranes represents a new opportunity to investigate the dynamic pathogenicity of the gut microbiome. The hypothesis that a decrease of sphingosine after I/R permits bacterial overgrowth in the intestine was not confirmed. © 2016 The Author(s) Published by S. Karger AG, Basel.

  16. My journey into the world of sphingolipids and sphingolipidoses

    PubMed Central

    SANDHOFF, Konrad

    2012-01-01

    Analysis of lipid storage in postmortem brains of patients with amaurotic idiocy led to the recognition of five lysosomal ganglioside storage diseases and identification of their inherited metabolic blocks. Purification of lysosomal acid sphingomyelinase and ceramidase and analysis of their gene structures were the prerequisites for the clarification of Niemann-Pick and Farber disease. For lipid catabolism, intraendosomal vesicles are formed during the endocytotic pathway. They are subjected to lipid sorting processes and were identified as luminal platforms for cellular lipid and membrane degradation. Lipid binding glycoproteins solubilize lipids from these cholesterol poor membranes and present them to water-soluble hydrolases for digestion. Biosynthesis and intracellular trafficking of lysosomal hydrolases (hexosaminidases, acid sphingomyelinase and ceramidase) and lipid binding and transfer proteins (GM2 activator, saposins) were analyzed to identify the molecular and metabolic basis of several sphingolipidoses. Studies on the biosynthesis of glycosphingolipids yielded the scheme of Combinatorial Ganglioside Biosynthesis involving promiscuous glycosyltransferases. Their defects in mutagenized mice impair brain development and function. PMID:23229750

  17. Lipid phosphate phosphatases regulate signal transduction through glycerolipids and sphingolipids.

    PubMed

    Brindley, David N; English, Denis; Pilquil, Carlos; Buri, Katherine; Ling, Zong Chao

    2002-05-23

    Lipid phosphate esters including lysophosphatidate (LPA), phosphatidate (PA), sphingosine 1-phosphate (S1P) and ceramide 1-phosphate (C1P) are bioactive in mammalian cells and serve as mediators of signal transduction. LPA and S1P are present in biological fluids and activate cells through stimulation of their respective G-protein-coupled receptors, LPA(1-3) and S1P(1-5). LPA stimulates fibroblast division and is important in wound repair. It is also active in maintaining the growth of ovarian cancers. S1P stimulates chemotaxis, proliferation and differentiation of vascular endothelial and smooth muscle cells and is an important participant in the angiogenic response and neovessel maturation. PA and C1P are believed to act primarily inside the cell where they facilitate vesicle transport. The lipid phosphates are substrates for a family of lipid phosphate phosphatases (LPPs) that dramatically alter the signaling balance between the phosphate esters and their dephosphorylated products. In the case of PA, S1P and C1P, the products are diacylglycerol (DAG), sphingosine and ceramide, respectively. These latter lipids are also bioactive and, thus, the LPPs change signals that the cell receives. The LPPs are integral membrane proteins that act both inside and outside the cell. The "ecto-activity" of the LPPs regulates the circulating and locally effective concentrations of LPA and S1P. Conversely, the internal activity controls the relative accumulation of PA or C1P in response to stimulation by various agonists thereby affecting cell signaling downstream of EDG and other receptors. This article will review the various LPPs and discuss how these enzymes could regulate signal transduction by lipid mediators.

  18. DNA damage response and sphingolipid signaling in liver diseases

    PubMed Central

    Matsuda, Yasunobu; Moro, Kazuki; Tsuchida, Junko; Soma, Daiki; Hirose, Yuki; Kobayashi, Takashi; Kosugi, Shin-ichi; Takabe, Kazuaki; Komatsu, Masaaki; Wakai, Toshifumi

    2016-01-01

    Patients with unresectable hepatocellular carcinoma (HCC) cannot generally be cured by systemic chemotherapy or radiotherapy due to their poor response to conventional therapeutic agents. The development of novel and efficient targeted therapies to increase their treatment options depends on the elucidation of the molecular mechanisms that underlie the pathogenesis of HCC. The DNA damage response (DDR) is a network of cell-signaling events that are triggered by DNA damage. Its dysregulation is thought to be one of the key mechanisms underlying the generation of HCC. Sphingosine-1-phosphate (S1P), a lipid mediator, has emerged as an important signaling molecule that has been found to be involved in many cellular functions. In the liver, the alteration of S1P signaling potentially affects the DDR pathways. In this review, we explore the role of the DDR in hepatocarcinogenesis of various etiologies, including hepatitis B and C infection and non-alcoholic steatohepatitis. Furthermore, we discuss the metabolism and functions of S1P that may affect the hepatic DDR. The elucidation of the pathogenic role of S1P may create new avenues of research into therapeutic strategies for patients with HCC. PMID:26514817

  19. Lipid phosphate phosphohydrolase-1 degrades exogenous glycerolipid and sphingolipid phosphate esters.

    PubMed Central

    Jasinska, R; Zhang, Q X; Pilquil, C; Singh, I; Xu, J; Dewald, J; Dillon, D A; Berthiaume, L G; Carman, G M; Waggoner, D W; Brindley, D N

    1999-01-01

    Lipid phosphate phosphohydrolase (LPP)-1 cDNA was cloned from a rat liver cDNA library. It codes for a 32-kDa protein that shares 87 and 82% amino acid sequence identities with putative products of murine and human LPP-1 cDNAs, respectively. Membrane fractions of rat2 fibroblasts that stably expressed mouse or rat LPP-1 exhibited 3.1-3. 6-fold higher specific activities for phosphatidate dephosphorylation compared with vector controls. Increases in the dephosphorylation of lysophosphatidate, ceramide 1-phosphate, sphingosine 1-phosphate and diacylglycerol pyrophosphate were similar to those for phosphatidate. Rat2 fibroblasts expressing mouse LPP-1 cDNA showed 1.6-2.3-fold increases in the hydrolysis of exogenous lysophosphatidate, phosphatidate and ceramide 1-phosphate compared with vector control cells. Recombinant LPP-1 was located partially in plasma membranes with its C-terminus on the cytosolic surface. Lysophosphatidate dephosphorylation was inhibited by extracellular Ca2+ and this inhibition was diminished by extracellular Mg2+. Changing intracellular Ca2+ concentrations did not alter exogenous lysophosphatidate dephosphorylation significantly. Permeabilized fibroblasts showed relatively little latency for the dephosphorylation of exogenous lysophosphatidate. LPP-1 expression decreased the activation of mitogen-activated protein kinase and DNA synthesis by exogenous lysophosphatidate. The product of LPP-1 cDNA is concluded to act partly to degrade exogenous lysophosphatidate and thereby regulate its effects on cell signalling. PMID:10359651

  20. Lipid phosphate phosphohydrolase-1 degrades exogenous glycerolipid and sphingolipid phosphate esters.

    PubMed

    Jasinska, R; Zhang, Q X; Pilquil, C; Singh, I; Xu, J; Dewald, J; Dillon, D A; Berthiaume, L G; Carman, G M; Waggoner, D W; Brindley, D N

    1999-06-15

    Lipid phosphate phosphohydrolase (LPP)-1 cDNA was cloned from a rat liver cDNA library. It codes for a 32-kDa protein that shares 87 and 82% amino acid sequence identities with putative products of murine and human LPP-1 cDNAs, respectively. Membrane fractions of rat2 fibroblasts that stably expressed mouse or rat LPP-1 exhibited 3.1-3. 6-fold higher specific activities for phosphatidate dephosphorylation compared with vector controls. Increases in the dephosphorylation of lysophosphatidate, ceramide 1-phosphate, sphingosine 1-phosphate and diacylglycerol pyrophosphate were similar to those for phosphatidate. Rat2 fibroblasts expressing mouse LPP-1 cDNA showed 1.6-2.3-fold increases in the hydrolysis of exogenous lysophosphatidate, phosphatidate and ceramide 1-phosphate compared with vector control cells. Recombinant LPP-1 was located partially in plasma membranes with its C-terminus on the cytosolic surface. Lysophosphatidate dephosphorylation was inhibited by extracellular Ca2+ and this inhibition was diminished by extracellular Mg2+. Changing intracellular Ca2+ concentrations did not alter exogenous lysophosphatidate dephosphorylation significantly. Permeabilized fibroblasts showed relatively little latency for the dephosphorylation of exogenous lysophosphatidate. LPP-1 expression decreased the activation of mitogen-activated protein kinase and DNA synthesis by exogenous lysophosphatidate. The product of LPP-1 cDNA is concluded to act partly to degrade exogenous lysophosphatidate and thereby regulate its effects on cell signalling.

  1. Switching head group selectivity in mammalian sphingolipid biosynthesis by active-site engineering of sphingomyelin synthases

    PubMed Central

    Kol, Matthijs; Panatala, Radhakrishnan; Nordmann, Mirjana; Swart, Leoni; van Suijlekom, Leonie; Cabukusta, Birol; Hilderink, Angelika; Grabietz, Tanja; Mina, John G. M.; Somerharju, Pentti; Korneev, Sergei; Tafesse, Fikadu G.; Holthuis, Joost C. M.

    2016-01-01

    SM is a fundamental component of mammalian cell membranes that contributes to mechanical stability, signaling, and sorting. Its production involves the transfer of phosphocholine from phosphatidylcholine onto ceramide, a reaction catalyzed by SM synthase (SMS) 1 in the Golgi and SMS2 at the plasma membrane. Mammalian cells also synthesize trace amounts of the SM analog ceramide phosphoethanolamine (CPE), but the physiological relevance of CPE production is unclear. Previous work revealed that SMS2 is a bifunctional enzyme producing both SM and CPE, whereas a closely related enzyme, sphingomyelin synthase-related protein (SMSr)/SAMD8, acts as a monofunctional CPE synthase in the endoplasmatic reticulum. Using domain swapping and site-directed mutagenesis on enzymes expressed in defined lipid environments, we here identified structural determinants that mediate head group selectivity of SMS family members. Notably, a single residue adjacent to the catalytic histidine in the third exoplasmic loop profoundly influenced enzyme specificity, with glutamic acid permitting SMS-catalyzed CPE production and aspartic acid confining the enzyme to produce SM. An exchange of exoplasmic residues with SMSr proved sufficient to convert SMS1 into a bulk CPE synthase. This allowed us to establish mammalian cells that produce CPE rather than SM as the principal phosphosphingolipid and provide a model of the molecular interactions that impart catalytic specificity among SMS enzymes. PMID:27165857

  2. Association of major histocompatibility complex II with cholesterol- and sphingolipid-rich membranes precedes peptide loading.

    PubMed

    Karacsonyi, Claudia; Knorr, Ruth; Fülbier, Angela; Lindner, Robert

    2004-08-13

    Major histocompatibility complex class II protein (MHC II) molecules present antigenic peptides to CD4-positive T-cells. Efficient T cell stimulation requires association of MHC II with membrane microdomains organized by cholesterol and glycosphingolipids or by tetraspanins. Using detergent extraction at 37 degrees C combined with a modified flotation assay, we investigated the sequence of events leading to the association of MHC II with cholesterol- and glycosphingolipid-rich membranes (DRMs) that are distinct from tetraspanins. We find two stages of association of MHC II with DRMs. In stage one, complexes of MHC II and invariant chain, a chaperone involved in MHC II transport, enter DRMs in the Golgi stack. In early endosomes, these complexes are almost quantitatively associated with DRMs. Upon transport to late endocytic compartments, MHC II-bound invariant chain is stepwise proteolyzed to the MHC class II-associated invariant chain peptide (CLIP) that remains MHC II-bound and retains a preference for DRMs. At the transition between the two stages, CLIP is exchanged against processed antigens, and the resulting MHC II-peptide complexes are transported to the cell surface. In the second stage, MHC II shows a lower overall association with DRMs. However, surface MHC II molecules occupied with peptides that induce resistance to denaturation by SDS are enriched in DRMs relative to SDS-sensitive MHC II-peptide complexes. Likewise, MHC II molecules loaded with long-lived processing products of hen-egg lysozyme containing the immunodominant epitope 48-61 show a very high preference for DRMs. Thus after an initial mainly intracellular stage of high DRM association, MHC II moves to a second stage in which its preference for DRMs is modulated by bound peptides.

  3. Structure of Sphingolipids From Sea Cucumber Cucumaria frondosa and Structure-Specific Cytotoxicity Against Human HepG2 Cells.

    PubMed

    Jia, Zicai; Song, Yu; Tao, Suyuan; Cong, Peixu; Wang, Xiaoxu; Xue, Changhu; Xu, Jie

    2016-03-01

    To investigate the relationship between structure and activity, three glucocerebroside series (CFC-1, CFC-2 and CFC-3), ceramides (CF-Cer) and long-chain bases (CF-LCB) of sea cucumber Cucumaria frondosa (C. frondosa) were isolated and evaluated in HepG2 cells. The molecular species of CFC-1, CFC-2 and CFC-3 and CF-Cer were identified using reversed-phase liquid chromatography with heated electrospray ionization coupled to high-resolution mass spectrometry (RPLC-HESI-HRMS), and determined on the basis of chemical and spectroscopic evidence: For the three glucocerebroside series, fatty acids (FA) were mainly saturated (18:0 and 22:0), monounsaturated (22:1, 23:1 and 24:1) and 2-hydroxyl FA (2-HFA) (23:1 h and 24:1 h), the structure of long-chain bases (LCB) were dihydroxy (d17:1, d18:1 and d18:2) and trihydroxy (t16:0 and t17:0), and the glycosylation was glucose; For CF-Cer, FA were primarily saturated (17:0) and monounsaturated (16:1 and 19:1), the structure of LCB were dihydroxy (d17:1 and d18:1), and trihydroxy (t16:0). The results of cell experiment indicated that all of three glucocerebroside series, CF-Cer and CF-LCB exhibited an inhibitory effects on cell proliferation. Moreover, CFC-3 was most effective in three glucocerebrosides to HepG-2 cell viability. The inhibition effect of CF-LCB was the strongest, and the inhibition effect of CF-Cer was much stronger than glucocerebrosides.

  4. Identification of a sphingolipid-specific phospholipase D activity associated with the generation of phytoceramide-1-phosphate in cabbage leaves.

    PubMed

    Tanaka, Tamotsu; Kida, Takashi; Imai, Hiroyuki; Morishige, Jun-ichi; Yamashita, Ryouhei; Matsuoka, Hisatsugu; Uozumi, Sachika; Satouchi, Kiyoshi; Nagano, Minoru; Tokumura, Akira

    2013-08-01

    The structure and biosynthetic route for an unidentified lipid (lipid X) detected by TLC of cabbage (Brassica oleracea) lipids was determined. Lipid X is a phospholipid that is resistant to mild alkali and detectable by MALDI-TOF MS as an adduct with Phos-tag, a phosphate-capture zinc complex. Various α-hydroxy fatty acids (16:0, 22:0, 24:0 and 24:1) were detected by GC-MS of fatty acid methyl esters prepared from lipid X. The deacyl derivative of lipid X was determined to be 4-hydroxysphingenine (dehydrophytosphingosine)-1-phosphate by MALDI-TOF MS with Phos-tag. From these results, lipid X was determined to be phytoceramide-1-phosphate (PC1P) with an α-hydroxy fatty acid. When cabbage homogenates were incubated, PC1P was formed, with a concomitant decrease in the amount of glycosylinositol phosphoceramide (GIPC). The formation of PC1P from GIPC was confirmed by treatment of purified cabbage GIPC with a membrane fraction of cabbage homogenates. Using a partially purified enzyme fraction, we found that the enzyme hydrolyzes GIPC specifically, but not glycerophospholipids and sphingomyelin. Arabidopsis thaliana also had this enzyme activity. From these results, we conclude that a previously uncharacterized phospholipase D activity that specifically hydrolyzes GIPC produces PC1P in brassicaceous plants. © 2013 FEBS.

  5. Stress-Induced Sphingolipid Signaling: Role of Type-2 Neutral Sphingomyelinase in Murine Cell Apoptosis and Proliferation

    PubMed Central

    Devillard, Raphael; Galvani, Sylvain; Thiers, Jean-Claude; Guenet, Jean-Louis; Hannun, Yusuf; Bielawski, Jacek; Nègre-Salvayre, Anne; Salvayre, Robert; Augé, Nathalie

    2010-01-01

    Background Sphingomyelin hydrolysis in response to stress-inducing agents, and subsequent ceramide generation, are implicated in various cellular responses, including apoptosis, inflammation and proliferation, depending on the nature of the different acidic or neutral sphingomyelinases. This study was carried out to investigate whether the neutral Mg2+-dependent neutral sphingomyelinase-2 (nSMase2) plays a role in the cellular signaling evoked by TNFalpha and oxidized LDLs, two stress-inducing agents, which are mitogenic at low concentrations and proapoptotic at higher concentrations. Methodology and Principal Findings For this purpose, we used nSMase2-deficient cells from homozygous fro/fro (fragilitas ossium) mice and nSMase2-deficient cells reconstituted with a V5-tagged nSMase2. We report that the genetic defect of nSMase2 (in fibroblasts from fro/fro mice) does not alter the TNFalpha and oxidized LDLs-mediated apoptotic response. Likewise, the hepatic toxicity of TNFalpha is similar in wild type and fro mice, thus is independent of nSMase2 activation. In contrast, the mitogenic response elicited by low concentrations of TNFalpha and oxidized LDLs (but not fetal calf serum) requires nSMase2 activation. Conclusion and Significance nSMase2 activation is not involved in apoptosis mediated by TNFalpha and oxidized LDLs in murine fibroblasts, and in the hepatotoxicity of TNFalpha in mice, but is required for the mitogenic response to stress-inducing agents. PMID:20352118

  6. Alteration of glycerolipid and sphingolipid-derived second messenger kinetics in ras transformed 3T3 cells.

    PubMed

    Laurenz, J C; Gunn, J M; Jolly, C A; Chapkin, R S

    1996-01-05

    The effect of ras transformation (rasB fibroblasts) on basal and serum-stimulated diacylglycerol (DAG) composition and mass was examined over time with respect to changes in membrane phospholipid composition and ceramide mass. RasB cells vs. nontransformed control cells (rasD and NR6) had chronically elevated DAG levels (up to 240 min) following serum stimulation, indicating a defect in the recovery phase of the intracellular DAG pulse. Ras transformation also had a dramatic effect on DAG composition. Molecular species analysis revealed that DAG from unstimulated rasB cells was enriched in the delta 9 desaturase fatty acyl species (monoenoate 18:1(n - 7) and 18:1(n - 9)), and depleted in arachidonic acid (20:4(n - 6)). With the exception of glycerophosphoinositol (GPI), DAG remodeling paralleled the compositional alterations in individual phospholipid classes. Importantly, ras transformation altered the fatty acyl composition of sphingomyelin, a precursor to the ceramide second messenger. With the addition of serum, control cells (rasD) had a progressive increase in ceramide mass with levels approximately 5-fold higher by 240 min. In contrast, ceramide levels did not increase in rasB cells at either 4 or 240 min. These results demonstrate that ras-oncogene, in addition to its effects on DAG metabolism, can also abolish the cellular increase in ceramide mass in response to serum stimulation. Since DAG and ceramide may have opposing biological functions, the prolonged elevation of DAG and the suppression of ceramide levels would be consistent with an enhanced proliferative capacity.

  7. Extrusion cooking with glucose supplementation of fumonsin- contaminated corn grits protects against nephrotoxicity and disrupted sphingolipid metabolism in rats

    USDA-ARS?s Scientific Manuscript database

    Fumonisins are mycotoxins produced by Fusarium verticillioides and F. proliferatum. They are found in corn and in corn-based foods. Fumonisin B1 (FB1), the most common fumonisin, causes animal diseases and, although its impact on human health is unclear, evidence suggests that it is a risk factor ...

  8. Lipid bilayers containing sphingomyelins and ceramides of varying N-acyl lengths: a glimpse into sphingolipid complexity.

    PubMed

    Jiménez-Rojo, Noemi; García-Arribas, Aritz B; Sot, Jesús; Alonso, Alicia; Goñi, Félix M

    2014-01-01

    The thermotropic properties of aqueous dispersions of sphingomyelins (SM) and ceramides (Cer) with N-acyl chains varying from C6:0 to C24:1, either pure or in binary mixtures, have been examined by differential scanning calorimetry. Even in the pure state, Cer and particularly SM exhibited complex endotherms, and their thermal properties did not vary in a predictable way with changes in structure. In some cases, e.g. C18:0 SM, atomic force microscopy revealed coexisting lamellar domains made of a single lipid. Partial chain interdigitation and metastable crystalline states were deemed responsible for the complex behavior. SM:Cer mixtures (90:10mol ratio) gave rise to bilayers containing separate SM-rich and Cer-rich domains. In vesicles made of more complex mixtures (SM:PE:Chol, 2:1:1), it is known that sphingomyelinase degradation of SM to Cer is accompanied by vesicle aggregation and release of aqueous contents. These vesicles did not reveal observable domain separation by confocal microscopy. Vesicle aggregation occurred at a faster rate for those bilayers that appeared to be more fluid according to differential scanning calorimetry. Content efflux rates measured by fluorescence spectroscopy were highest with C18:0 and C18:1 SM, and in general those rates did not vary regularly with other physical properties of SM or Cer. In general the individual SM and Cer appear to have particular thermotropic properties, often unrelated to the changes in N-acyl chain.

  9. Combined gas chromatography-chemical ionization mass spectrometry of sphingolipids. I. Glucosyl sphingosine, ceramides and cerebrosides of the spleen in Gaucher's disease.

    PubMed

    Oshima, M; Ariga, T; Murata, T

    1977-08-01

    Trimethylsilylated glucosyl sphingosine, ceramides and glucocerebrosides were analysed by combined gas chromatography (GC)-chemical ionization (CI) mass spectrometry. Isobutane, methane and ammonia were used as reactant gasses for chemical ionization. Essentially the same fragment ions were detected in the spectra with the different reactant gases. Purified glucocerebrosides isolated from the spleen of a patient with Gaucher's disease were clearly separated into their 8 molecular species by gas chromatography. Three other minor components were detected by spectrometry, and these 11 molecular species of glucocerbrosides from the spleen of the patient with Gaucher's disease have been analysed. The ceramides obtained by periodate oxidation and then alkaline reduction of the glucocerebrosides were also separated into 11 molecular species by GC-CI mass spectrometry. Molecular weight could be determined using the major fragment ion of m/e (M+-90) in the spectra of ceramides and cerebrosides. The chemical ionization method is useful for structural analysis and determination of the molecular species of sphingoglycolipids.

  10. An efficient preparation of isosteric phosphonate analogues of sphingolipids by opening of oxirane and cyclic sulfamidate intermediates with alpha-lithiated alkylphosphonic esters.

    PubMed

    Sun, Chaode; Bittman, Robert

    2004-10-29

    D-erythro-(2S,3R,4E)-Sphingosine-1-phosphonate (1), the isosteric phosphonate analogue of naturally occurring sphingosine 1-phosphate (1a), and D-ribo-phytosphingosine 1-phosphonate (2), the isosteric phosphonate analogue of D-ribo-phytosphingosine-1-phosphate (2a), were synthesized starting with methyl 2,3-O-isopropylidene-d-glycerate (4) and D-ribo-phytosphingosine (3), respectively. Oxirane 12 was formed in eight steps from 4, and cyclic sulfamidate 22 was formed in five steps from 3. The phosphonate group was introduced via regioselective ring-opening reactions of oxirane 12 and cyclic sulfamidate 22 with lithium dialkyl methylphosphonate, affording 13 and 23, respectively. The synthesis of 1 was completed by S(N)2 displacement of chloromesylate intermediate 14b with azide ion, followed by conversion of the resulting azido group to a NHBoc group and deprotection. The synthesis of 2 was completed by cleavage of the acetal, N-benzyl, and alkyl phosphonate ester groups.

  11. Non-target analysis of phospholipid and sphingolipid species in egg yolk using liquid chromatography/triple quadrupole tandem mass spectrometry.

    PubMed

    Buszewski, Bogusław; Walczak, Justyna; Žuvela, Petar; Liu, J Jay

    2017-03-03

    In this work, phospholipids extracted from egg yolk (control group, experimental group) were identified using high performance liquid chromatography coupled with electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS). Combinations of fatty acyls occurring in 11 classes of phospholipids from egg yolk were investigated. Differences between the profile of fatty acyls from hens fed traditionally and the ones that received special diet supplementation were observed. Experimental findings were complemented with multivariate chemometric analysis. Multiple reaction monitoring mass spectrometry mode was utilized and 123 distinct combinations of fatty acyls occurring in phospholipids were identified. From these, large portions are polyunsaturated fatty acyls from the omega-3 and omega-6 family. HPLC MS/MS analysis allows for quick, accurate and precise determination of biologically active compounds, found in low concentrations within the tested material. Copyright © 2017. Published by Elsevier B.V.

  12. A novel fluorescent ceramide analogue for studying membrane traffic in animal cells: accumulation at the Golgi apparatus results in altered spectral properties of the sphingolipid precursor

    PubMed Central

    1991-01-01

    A series of ceramide analogues bearing the fluorophore boron dipyrromethene difluoride (BODIPY) were synthesized and evaluated as vital stains for the Golgi apparatus, and as tools for studying lipid traffic between the Golgi apparatus and the plasma membrane of living cells. Studies of the spectral properties of several of the BODIPY- labeled ceramides in lipid vesicles demonstrated that the fluorescence emission maxima were strongly dependent upon the molar density of the probes in the membrane. This was especially evident using N-[5-(5,7- dimethyl BODIPY)-1-pentanoyl]-D-erythro-sphingosine (C5-DMB-Cer), which exhibited a shift in its emission maximum from green (integral of 515 nm) to red (integral of 620 nm) wavelengths with increasing concentrations. When C5-DMB-Cer was used to label living cells, this property allowed us to differentiate membranes containing high concentrations of the fluorescent lipid and its metabolites (the corresponding analogues of sphingomyelin and glucosylceramide) from other regions of the cell where smaller amounts of the probe were present. Using this approach, prominent red fluorescent labeling of the Golgi apparatus, Golgi apparatus-associated tubulovesicular processes, and putative Golgi apparatus transport vesicles was seen in living human skin fibroblasts, as well as in other cell types. Based on fluorescence ratio imaging microscopy, we estimate that C5-DMB-Cer and its metabolites were present in Golgi apparatus membranes at concentrations up to 5-10 mol %. In addition, the concentration- dependent spectral properties of C5-DMB-Cer were used to monitor the transport of C5-DMB-lipids to the cell surface at 37 degrees C. PMID:2045412

  13. A stereocontrolled, efficient synthetic route to bioactive sphingolipids: synthesis of phytosphingosine and phytoceramides from unsaturated ester precursors via cyclic sulfate intermediates.

    PubMed

    He, L; Byun, H S; Bittman, R

    2000-11-03

    An efficient and highly enantioselective method for the preparation of D-ribo- and L-lyxo-phytosphingosines (1a,b, respectively) and phytoceramides (2a,b) has been developed. The key steps in the syntheses are as follows: (i) osmium-catalyzed asymmetric dihydroxylation of 4-O-protected (E)-alpha,beta-unsaturated ester 5 (generated by dihydroxylation of 1-hexadecene, followed by oxidation to the aldehyde and Horner-Wadsworth-Emmons olefination), (ii) conversion to cyclic sulfate intermediate 7, and (iii) regioselective alpha-azidation of 7. Reduction of 4-O-protected 2-azido ester 8 via alpha-azidolactone 9 afforded phytosphingosine 1a. Staudinger reduction of the azido group of 8, followed by in situ N-acylation in aqueous media and reduction of the ester functionality with NaBH(4)/LiBr, provided phytoceramide 2a. By using a similar approach, phytosphingosine 1b was synthesized. D-erythro-4, 5-Dihydrosphingosine 1c and D-erythro-4,5-dihydroceramide 2c were synthesized in high yield from 1-hexadecanol via cyclic sulfate intermediate 15. The desired configurations at C-2, C-3, and C-4 of the sphingoid chain can be accessed readily by the route described here.

  14. Gastrointestinal Degradation of Fumonisin B1 by Carboxylesterase FumD Prevents Fumonisin Induced Alteration of Sphingolipid Metabolism in Turkey and Swine

    PubMed Central

    Masching, Sabine; Naehrer, Karin; Schwartz-Zimmermann, Heidi-Elisabeth; Sărăndan, Mihai; Schaumberger, Simone; Dohnal, Ilse; Nagl, Veronika; Schatzmayr, Dian

    2016-01-01

    The mycotoxin fumonisin B1 (FB1) is a frequent contaminant of feed and causes various adverse health effects in domestic animals. Hence, effective strategies are needed to prevent the impact of fumonisins on livestock productivity. Here we evaluated the capability of the fumonisin carboxylesterase FumD to degrade FB1 to its less toxic metabolite hydrolyzed FB1 (HFB1) in the gastrointestinal tract of turkeys and pigs. First, an ex vivo pig model was used to examine the activity of FumD under digestive conditions. Within 2 h of incubation with FumD, FB1 was completely degraded to HFB1 in the duodenum and jejunum, respectively. To test the efficacy of the commercial application of FumD (FUMzyme) in vivo, female turkeys (n = 5) received either basal feed (CON), fumonisin-contaminated feed (15 mg/kg FB1+FB2; FB) or fumonisin-contaminated feed supplemented with FUMzyme (15 U/kg; FB+FUMzyme) for 14 days ad libitum. Addition of FUMzyme resulted in significantly decreased levels of FB1 in excreta, whereas HFB1 concentrations were significantly increased. Compared to the FB group (0.24 ± 0.02), the mean serum sphinganine-to-sphingosine (Sa/So) ratio was significantly reduced in the FB+FUMzyme group (0.19 ± 0.02), thus resembling values of the CON group (0.16 ± 0.02). Similarly, exposure of piglets (n = 10) to 2 mg/kg FB1+FB2 for 42 days caused significantly elevated serum Sa/So ratios (0.39 ± 0.15) compared to the CON group (0.14 ± 0.01). Supplementation with FUMzyme (60 U/kg) resulted in gastrointestinal degradation of FB1 and unaffected Sa/So ratios (0.16 ± 0.02). Thus, the carboxylesterase FumD represents an effective strategy to detoxify FB1 in the digestive tract of turkeys and pigs. PMID:27007395

  15. Effect of high fat diet enriched with unsaturated and diet rich in saturated fatty acids on sphingolipid metabolism in rat skeletal muscle.

    PubMed

    Blachnio-Zabielska, Agnieszka; Baranowski, Marcin; Zabielski, Piotr; Gorski, Jan

    2010-11-01

    Consumption of high fat diet leads to muscle lipid accumulation which is an important factor involved in induction of insulin resistance. Ceramide is likely to partially inhibit insulin signaling cascade. The aim of this study was to examine the effect of different high fat diets on ceramide metabolism in rat skeletal muscles. The experiments were carried out on rats fed for 5 weeks: (1) a standard chow and (2) high fat diet rich in polyunsaturated fatty acids (PUFA) and (3) diet enriched with saturated fatty acids (SAT). Assays were performed on three types of muscles: slow-twitch oxidative (soleus), fast-twitch oxidative-glycolytic, and fast-twitch glycolytic (red and white section of the gastrocnemius, respectively). The activity of serine palmitoyltransferase (SPT), neutral and acid sphingomyelinase (n- and aSMase), and neutral and alkaline ceramidase (n- and alCDase) was examined. The content of ceramide, sphinganine, sphingosine, and sphingosine-1-phosphate was also measured. The ceramide content did not change in any muscle from PUFA diet group but increased in the SAT diet group by 46% and 52% in the soleus and red section of the gastrocnemius, respectively. Elevated ceramide content in the SAT diet group could be a result of increased SPT activity and simultaneously decreased activity of nCDase. Unchanged ceramide content in the PUFA diet group might be a result of increased activity of SPT and alCDase and simultaneously decreased activity of SMases. We conclude that regulation of muscle ceramide level depends on the diet and type of skeletal muscle. © 2010 Wiley-Liss, Inc.

  16. Membrane Phosphoproteomics of Yeast Early Response to Acetic Acid: Role of Hrk1 Kinase and Lipid Biosynthetic Pathways, in Particular Sphingolipids

    PubMed Central

    Guerreiro, Joana F.; Mira, Nuno P.; Santos, Aline X. S.; Riezman, Howard; Sá-Correia, Isabel

    2017-01-01

    Saccharomyces cerevisiae response and tolerance to acetic acid is critical in industrial biotechnology and in acidic food and beverages preservation. The HRK1 gene, encoding a protein kinase of unknown function belonging to the “Npr1-family” of kinases known to be involved in the regulation of plasma membrane transporters, is an important determinant of acetic acid tolerance. This study was performed to identify the alterations occurring in yeast membrane phosphoproteome profile during the adaptive early response to acetic acid stress (following 1 h of exposure to a sub-lethal inhibitory concentration; 50 mM at pH 4.0) and the effect of HRK1 expression on the phosphoproteome. Results from mass spectrometry analysis following the prefractionation and specific enrichment of phosphorylated peptides using TiO2 beads highlight the contribution of processes related with translation, protein folding and processing, transport, and cellular homeostasis in yeast response to acetic acid stress, with particular relevance for changes in phosphorylation of transport-related proteins, found to be highly dependent on the Hrk1 kinase. Twenty different phosphoproteins known to be involved in lipid and sterol metabolism were found to be differently phosphorylated in response to acetic acid stress, including several phosphopeptides that had not previously been described as being phosphorylated. The suggested occurrence of cellular lipid composition remodeling during the short term yeast response to acetic acid was confirmed: Hrk1 kinase-independent reduction in phytoceramide levels and a reduction in phosphatidylcholine and phosphatidylinositol levels under acetic acid stress in the more susceptible hrk1Δ strain were revealed by a lipidomic analysis. PMID:28747907

  17. Membrane Phosphoproteomics of Yeast Early Response to Acetic Acid: Role of Hrk1 Kinase and Lipid Biosynthetic Pathways, in Particular Sphingolipids.

    PubMed

    Guerreiro, Joana F; Mira, Nuno P; Santos, Aline X S; Riezman, Howard; Sá-Correia, Isabel

    2017-01-01

    Saccharomyces cerevisiae response and tolerance to acetic acid is critical in industrial biotechnology and in acidic food and beverages preservation. The HRK1 gene, encoding a protein kinase of unknown function belonging to the "Npr1-family" of kinases known to be involved in the regulation of plasma membrane transporters, is an important determinant of acetic acid tolerance. This study was performed to identify the alterations occurring in yeast membrane phosphoproteome profile during the adaptive early response to acetic acid stress (following 1 h of exposure to a sub-lethal inhibitory concentration; 50 mM at pH 4.0) and the effect of HRK1 expression on the phosphoproteome. Results from mass spectrometry analysis following the prefractionation and specific enrichment of phosphorylated peptides using TiO2 beads highlight the contribution of processes related with translation, protein folding and processing, transport, and cellular homeostasis in yeast response to acetic acid stress, with particular relevance for changes in phosphorylation of transport-related proteins, found to be highly dependent on the Hrk1 kinase. Twenty different phosphoproteins known to be involved in lipid and sterol metabolism were found to be differently phosphorylated in response to acetic acid stress, including several phosphopeptides that had not previously been described as being phosphorylated. The suggested occurrence of cellular lipid composition remodeling during the short term yeast response to acetic acid was confirmed: Hrk1 kinase-independent reduction in phytoceramide levels and a reduction in phosphatidylcholine and phosphatidylinositol levels under acetic acid stress in the more susceptible hrk1Δ strain were revealed by a lipidomic analysis.

  18. Gastrointestinal Degradation of Fumonisin B₁ by Carboxylesterase FumD Prevents Fumonisin Induced Alteration of Sphingolipid Metabolism in Turkey and Swine.

    PubMed

    Masching, Sabine; Naehrer, Karin; Schwartz-Zimmermann, Heidi-Elisabeth; Sărăndan, Mihai; Schaumberger, Simone; Dohnal, Ilse; Nagl, Veronika; Schatzmayr, Dian

    2016-03-21

    The mycotoxin fumonisin B₁ (FB₁) is a frequent contaminant of feed and causes various adverse health effects in domestic animals. Hence, effective strategies are needed to prevent the impact of fumonisins on livestock productivity. Here we evaluated the capability of the fumonisin carboxylesterase FumD to degrade FB₁ to its less toxic metabolite hydrolyzed FB₁ (HFB₁) in the gastrointestinal tract of turkeys and pigs. First, an ex vivo pig model was used to examine the activity of FumD under digestive conditions. Within 2 h of incubation with FumD, FB₁ was completely degraded to HFB₁ in the duodenum and jejunum, respectively. To test the efficacy of the commercial application of FumD (FUMzyme) in vivo, female turkeys (n = 5) received either basal feed (CON), fumonisin-contaminated feed (15 mg/kg FB₁+FB₂; FB) or fumonisin-contaminated feed supplemented with FUMzyme (15 U/kg; FB+FUMzyme) for 14 days ad libitum. Addition of FUMzyme resulted in significantly decreased levels of FB₁ in excreta, whereas HFB₁ concentrations were significantly increased. Compared to the FB group (0.24 ± 0.02), the mean serum sphinganine-to-sphingosine (Sa/So) ratio was significantly reduced in the FB+FUMzyme group (0.19 ± 0.02), thus resembling values of the CON group (0.16 ± 0.02). Similarly, exposure of piglets (n = 10) to 2 mg/kg FB₁+FB₂ for 42 days caused significantly elevated serum Sa/So ratios (0.39 ± 0.15) compared to the CON group (0.14 ± 0.01). Supplementation with FUMzyme (60 U/kg) resulted in gastrointestinal degradation of FB₁ and unaffected Sa/So ratios (0.16 ± 0.02). Thus, the carboxylesterase FumD represents an effective strategy to detoxify FB₁ in the digestive tract of turkeys and pigs.

  19. Identification of Two Suppressors of CSG2 Calcium Sensitivity, SCS7 and SUR2, as Genes Encoding Hydroxylases of the Sphingolipid Biosynthetic Pathway of Saccharomyces cerevisiae

    DTIC Science & Technology

    1997-12-10

    pyridoxyl phosphate enzyme likely to catalyze condensation between the a-carbon ofan amino acid and the carbonyl group ofan acyl-CoA. SCSI was found...analysis is YMR272C on the right arm ofChromosome XIII. It contains 1155 base pairs predicted to encode a 385 amino acid protein. In order to gain insight...niacin, pyri- doxine HCl, thiamine Hel, and MnC12; and 0.2 Jlg/ml each ofFeC13·6H20 and amino benzoic acid . Nucleic Acid Manipulation. Southern Blot

  20. A Blood Spot Method for Detecting Fumonisin-Induced Changes in Putative Sphingolipid Biomarkers in LM/Bc Mice and Humans

    USDA-ARS?s Scientific Manuscript database

    Fumonisin B1 (FB1) is a toxic chemical produced by molds. The molds that produce fumonisin are common in corn. Consumption of contaminated corn by farm animals has been shown to be the cause of disease. Fumonisin has been hypothesized to be an environmental risk factor for diseases in humans in c...

  1. Sphingolipid bases and their 1-phosphates, but not fumonisins, are translocated from roots to aerial tissues of maize seedlings watered with fumonisins.

    USDA-ARS?s Scientific Manuscript database

    In an earlier study using maize seedlings grown from kernels inoculated with Fusarium verticillioides, fumonisin B1 (FB1) was preferentially accumulated in leaf tissue compared to FB2 and FB3. The present study tested whether maize seedlings preferentially translocate FB1 when plants are watered wit...

  2. A neutral sphingomyelinase resides in sphingolipid-enriched microdomains and is inhibited by the caveolin-scaffolding domain: potential implications in tumour necrosis factor signalling.

    PubMed Central

    Veldman, R J; Maestre, N; Aduib, O M; Medin, J A; Salvayre, R; Levade, T

    2001-01-01

    Sphingomyelinases hydrolyse sphingomyelin to ceramide, a process involved in signal-transduction routes leading to apoptosis and various other cellular responses. In the present study, we investigated the sphingomyelinase content of caveolae, invaginated plasma-membrane microdomains that contain a variety of signalling molecules. These structures are highly enriched in sphingomyelin as well as in ceramide, which suggests that metabolism of these lipids might, to some extent, occur locally. By cell fractionation, we demonstrate that, in addition to a previously reported minute amount of acidic sphingomyelinase activity, a substantial amount of neutral sphingomyelinase activity resides in caveolae of human skin fibroblasts. This caveolar neutral sphingomyelinase activity was also detected in Niemann-Pick disease type A fibroblasts, which are completely devoid of functional acidic sphingomyelinase. Neutral (but not acidic) sphingomyelinase activity was specifically inhibited by a peptide that corresponds to the scaffolding domain of caveolin, which suggests a direct molecular interaction between the two proteins. In addition, this finding implies a cytosolic orientation of the caveolar neutral sphingomyelinase. Interestingly, stimulation of fibroblasts with tumour necrosis factor alpha (TNFalpha) resulted in a partial shift of its p55 receptor to caveolin-enriched membrane fractions and the appearance of caveolin-sensitive neutral sphingomyelinase activity in the non-caveolar fractions. These results suggest that (part of) the presently identified caveolar neutral sphingomyelinase activity is involved in TNFalpha signalling. PMID:11311151

  3. Direct interaction between the inhibitor 2 and ceramide via sphingolipid-protein binding is involved in the regulation of protein phosphatase 2A activity and signaling.

    PubMed

    Mukhopadhyay, Archana; Saddoughi, Sahar A; Song, Pengfei; Sultan, Iyad; Ponnusamy, Suriyan; Senkal, Can E; Snook, Christopher F; Arnold, Hugh K; Sears, Rosalie C; Hannun, Yusuf A; Ogretmen, Besim

    2009-03-01

    In this study, the inhibitor 2 of protein phosphatase 2A (I2PP2A) was identified in vitro and in situ as a ceramide-binding protein, which exhibits stereoisomer specificity and fatty acid chain length preference. Site- directed mutagenesis coupled with structural details of I2PP2A suggested that VIK 207-209 residues localized on helix 7 are important for ceramide binding and single mutation of K209D altered this interaction. Notably, I2PP2A-ceramide binding decreased the association between PP2A and the inhibitor, preventing the inhibition of PP2A activity in vitro. In addition, studies in A549 human lung cancer cells revealed that ceramide mediates c-Myc degradation via its PP2A-dependent dephosphorylation at S62, and treatment with okadaic acid and expression of c-Myc mutants with S62A or S62D conversions resulted in resistance to ceramide-mediated degradation. Importantly, whereas down-regulation of I2PP2A enhanced PP2A-mediated c-Myc degradation in response to ceramide, ectopic expression of wild-type I2PP2A but not of its K209D mutant protected this degradation in A549 cells. Moreover, expression of wild-type I2PP2A prevented the growth-inhibitory effects of ceramide both against A549 cells and xenograft-driven tumors in situ and in vivo compared with that in controls. Thus, these results suggest that direct interaction of I2PP2A with ceramide plays important biological roles via the regulation of PP2A activity and signaling, which in turn control ceramide-mediated degradation of c-Myc and antiproliferation.

  4. Binding of the sphingolipid S1P to hTERT stabilizes telomerase at the nuclear periphery by allosterically mimicking protein phosphorylation.

    PubMed

    Panneer Selvam, Shanmugam; De Palma, Ryan M; Oaks, Joshua J; Oleinik, Natalia; Peterson, Yuri K; Stahelin, Robert V; Skordalakes, Emmanuel; Ponnusamy, Suriyan; Garrett-Mayer, Elizabeth; Smith, Charles D; Ogretmen, Besim

    2015-06-16

    During DNA replication, the enzyme telomerase maintains the ends of chromosomes, called telomeres. Shortened telomeres trigger cell senescence, and cancer cells often have increased telomerase activity to promote their ability to proliferate indefinitely. The catalytic subunit, human telomerase reverse transcriptase (hTERT), is stabilized by phosphorylation. We found that the lysophospholipid sphingosine 1-phosphate (S1P), generated by sphingosine kinase 2 (SK2), bound hTERT at the nuclear periphery in human and mouse fibroblasts. Docking predictions and mutational analyses revealed that binding occurred between a hydroxyl group (C'3-OH) in S1P and Asp(684) in hTERT. Inhibiting or depleting SK2 or mutating the S1P binding site decreased the stability of hTERT in cultured cells and promoted senescence and loss of telomere integrity. S1P binding inhibited the interaction of hTERT with makorin ring finger protein 1 (MKRN1), an E3 ubiquitin ligase that tags hTERT for degradation. Murine Lewis lung carcinoma (LLC) cells formed smaller tumors in mice lacking SK2 than in wild-type mice, and knocking down SK2 in LLC cells before implantation into mice suppressed their growth. Pharmacologically inhibiting SK2 decreased the growth of subcutaneous A549 lung cancer cell-derived xenografts in mice, and expression of wild-type hTERT, but not an S1P-binding mutant, restored tumor growth. Thus, our data suggest that S1P binding to hTERT allosterically mimicks phosphorylation, promoting telomerase stability and hence telomere maintenance, cell proliferation, and tumor growth.

  5. Mitochondrial protein alterations in a familial peripheral neuropathy caused by the V144D amino acid mutation in the sphingolipid protein, SPTLC1.

    PubMed

    Stimpson, Scott E; Coorssen, Jens R; Myers, Simon J

    2015-01-01

    Axonal degeneration is the final common path in many neurological disorders. Subsets of neuropathies involving the sensory neuron are known as hereditary sensory neuropathies (HSNs). Hereditary sensory neuropathy type I (HSN-I) is the most common subtype of HSN with autosomal dominant inheritance. It is characterized by the progressive degeneration of the dorsal root ganglion (DRG) with clinical symptom onset between the second or third decade of life. Heterozygous mutations in the serine palmitoyltransferase (SPT) long chain subunit 1 (SPTLC1) gene were identified as the pathogenic cause of HSN-I. Ultrastructural analysis of mitochondria from HSN-I patient cells has displayed unique morphological abnormalities that are clustered to the perinucleus where they are wrapped by the endoplasmic reticulum (ER). This investigation defines a small subset of proteins with major alterations in abundance in mitochondria harvested from HSN-I mutant SPTLC1 cells. Using mitochondrial protein isolates from control and patient lymphoblasts, and a combination of 2D gel electrophoresis, immunoblotting and mass spectrometry, we have shown the increased abundance of ubiquinol-cytochrome c reductase core protein 1, an electron transport chain protein, as well as the immunoglobulin, Ig kappa chain C. The regulation of these proteins may provide a new route to understanding the cellular and molecular mechanisms underlying HSN-I.

  6. Suppressors (scsl-scs7) of CSG2, a Gene Required by Saccharomyces cerevisiae for Growth in Media Containing 10 mMCa(2+), Identify Genes Required for Sphingolipid Biosynthesis

    DTIC Science & Technology

    1994-07-01

    activity and requires phytosphingosine or dihydrosphingarune for growth indicating that SCSI encodes serine palmitoyltransferase. This enzyme... phytosphingosine on the growth rate of scsI csg2, scsI CSG2, scsI::LEUZ’ CSGZ, SCSI csgZ and SCSI CSGZ. 87 20. Acid titration of wild·type and scsI-I cells. 21...ceramide) and a polar head group. The ceramide consists of a long-chain base (sphingosine in animals, phytosphingosine (PHS) in fungi and plants) and a

  7. Analysis of sphingolipids in potatoes (Solanum tuberosum L.) and sweet potatoes (Ipomoea batatas (L.) Lam.) by reversed phase high-performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS).

    PubMed

    Bartke, Nana; Fischbeck, Anne; Humpf, Hans-Ulrich

    2006-12-01

    Ceramides and glucocerebrosides of potatoes (Solanum tuberosum L.) and sweet potatoes (Ipomoea batatas (L.) Lam.) were analyzed using RP-HPLC-ESI-MS/MS. Ceramides and glucocerebrosides containing the three different long-chain bases 4,8-sphingadienine (d18:2(delta4,delta8)), 4-hydroxy-8-sphingenine (t18:1(delta8)), and 8-sphingenine (d18:1(delta8)) acylated to saturated and unsaturated hydroxy- and nonhydroxy fatty acids with 16-26 carbon atoms were detected. For ceramides and glucocerebrosides 4,8-sphingadienine (d18:2(delta4,delta8)) was found as the major long-chain base, with lesser amounts of 4-hydroxy-8-sphingenine (t18:1(delta8)) and 8-sphingenine (d18:1(delta8)). 2-(Alpha-)hydroxypalmitic acid (C16:0h) was the major fatty acid, which was found to be acylated to the long-chain bases. For quantification of these compounds, an RP-HPLC-ESI-MS/MS method with an "echo-peak"-technique simulating internal standard injection was developed. The analyzed samples of potatoes and sweet potatoes showed amounts of approximately 0.1-8 microg/kg single ceramides and amounts up to 500 microg/kg glucocerebrosides, with C16:0h-glucosyl-4,8-sphingadienine as the major component.

  8. Cognitive status and vitamin K status in older men and women

    USDA-ARS?s Scientific Manuscript database

    Vitamin K has been implicated in the regulation of sphingolipid metabolism. In rats, vitamin K deficiency decreases sphingolipid concentrations, resulting in changes in behavior, but not short-term memory. The association between vitamin K intake and cognition in humans is not well studied. In 379 m...

  9. C16:0-Ceramide Signals Insulin Resistance

    PubMed Central

    Hla, Timothy; Kolesnick, Richard

    2015-01-01

    A substantive literature has accumulated implicating sphingolipids, in particular ceramides, as mediators of insulin resistance in metabolic syndrome. Thanks to recent technical advances in mouse genetics and lipidomics, two independent laboratories identify the same sphingolipid, C16:0-ceramide, as principal mediator of obesity-related insulin resistance. PMID:25440051

  10. Comparative plant sphingolipidomic reveals specific lipids in seeds and oil.

    PubMed

    Tellier, Frédérique; Maia-Grondard, Alessandra; Schmitz-Afonso, Isabelle; Faure, Jean-Denis

    2014-07-01

    Plant sphingolipids are a highly diverse family of structural and signal lipids. Owing to their chemical diversity and complexity, a powerful analytical method was required to identify and quantify a large number of individual molecules with a high degree of structural accuracy. By using ultra-performance liquid chromatography with a single elution system coupled to electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) in the positive multiple reaction monitoring (MRM) mode, detailed sphingolipid composition was analyzed in various tissues of two Brassicaceae species Arabidopsis thaliana and Camelina sativa. A total of 300 molecular species were identified defining nine classes of sphingolipids, including Cers, hCers, Glcs and GIPCs. High-resolution mass spectrometry identified sphingolipids including amino- and N-acylated-GIPCs. The comparative analysis of seedling, seed and oil sphingolipids showed tissue specific distribution suggesting metabolic channeling and compartmentalization. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. Activation of 3-Phosphoinositide-dependent Kinase 1 (PDK1) and Serum- and Glucocorticoid-induced Protein Kinase 1 (SGK1) by Short-chain Sphingolipid C4-ceramide Rescues the Trafficking Defect of ΔF508-Cystic Fibrosis Transmembrane Conductance Regulator (ΔF508-CFTR)*

    PubMed Central

    Caohuy, Hung; Yang, Qingfeng; Eudy, Yvonne; Ha, Thien-An; Xu, Andrew E.; Glover, Matthew; Frizzell, Raymond A.; Jozwik, Catherine; Pollard, Harvey B.

    2014-01-01

    Cystic fibrosis (CF) is due to a folding defect in the CF transmembrane conductance regulator (CFTR) protein. The most common mutation, ΔF508, prevents CFTR from trafficking to the apical plasma membrane. Here we show that activation of the PDK1/SGK1 signaling pathway with C4-ceramide (C4-CER), a non-toxic small molecule, functionally corrects the trafficking defect in both cultured CF cells and primary epithelial cell explants from CF patients. The mechanism of C4-CER action involves a series of mutual autophosphorylation and phosphorylation events between PDK1 and SGK1. Detailed mechanistic studies indicate that C4-CER initially induces autophosphorylation of SGK1 at Ser422. SGK1[Ser(P)422] and C4-CER coincidently bind PDK1 and permit PDK1 to autophosphorylate at Ser241. Then PDK1[Ser(P)241] phosphorylates SGK1[Ser(P)422] at Thr256 to generate fully activated SGK1[Ser422, Thr(P)256]. SGK1[Ser(P)422,Thr(P)256] phosphorylates and inactivates the E3 ubiquitin ligase Nedd4-2. ΔF508-CFTR is thus free to traffic to the plasma membrane. Importantly, C4-CER-mediated activation of both PDK1 and SGK1 is independent of the PI3K/Akt/mammalian target of rapamycin signaling pathway. Physiologically, C4-CER significantly increases maturation and stability of ΔF508-CFTR (t½ ∼10 h), enhances cAMP-activated chloride secretion, and suppresses hypersecretion of interleukin-8 (IL-8). We suggest that candidate drugs for CF directed against the PDK1/SGK1 signaling pathway, such as C4-CER, provide a novel therapeutic strategy for a life-limiting disorder that affects one child, on average, each day. PMID:25384981

  12. Introduction to tools and techniques for ceramide-centered research.

    PubMed

    Kitatani, Kazuyuki; Luberto, Chiara

    2010-01-01

    Sphingolipids are important components of eukaryotic cells, many of which function as bioactive signaling molecules. As thoroughly discussed elsewhere in this volume, ceramide, central metabolite of the sphingolipid pathway, plays key roles in a variety of cellular responses. Since the discovery of the bioactive function of ceramide, a growing number of tools and techniques have been and still are being developed in order to better decipher the complexity and implications of ceramide-mediated signaling. With this chapter it is our intention to provide new comers to the sphingolipid arena with a short overview of tools and techniques currently available for the study ofsphingolipid metabolism, with the focus on ceramide.

  13. Tree nut phytochemicals: composition, antioxidant capacity, bioactivity, impact factors. A systematic review of almonds, Brazils, cashews, hazelnuts, macadamias, pecans, pine nuts, pistachios and walnuts

    USDA-ARS?s Scientific Manuscript database

    Tree nuts contain an array of phytochemicals including carotenoids, phenolic acids, phytosterols and polyphenolic compounds such as flavonoids, proanthocyanidins (PAC) and stilbenes, all of which are included in nutrient databases, as well as phytates, sphingolipids, alkylphenols and lignans, which ...

  14. Ceramide induced mitophagy and tumor suppression

    PubMed Central

    Dany, Mohammed; Ogretmen, Besim

    2015-01-01

    Sphingolipids are bioactive lipid effectors, which are involved in the regulation of various cellular signaling pathways. Sphingolipids play essential roles in controlling cell inflammation, proliferation, death, migration, senescence, metastasis and autophagy. Alterations in sphingolipid metabolism has been also implicated in many human cancers. Macroautophagy (referred to here as autophagy) is a form of nonselective sequestering of cytosolic materials by double membrane structures, autophagosomes, which can be either protective or lethal for cells. Ceramide, a central molecule of sphingolipid metabolism is involved in the regulation of autophagy at various levels, including the induction of lethal mitophagy, a selective autophagy process to target and eliminate damaged mitochondria. In this review, we focused on recent studies with regard to the regulation of autophagy, in particular lethal mitophagy, by ceramide, and aimed at providing discussion points for various context-dependent roles and mechanisms of action of ceramide in controlling mitophagy. PMID:25634657

  15. Sphingoid Bases and the Serine Catabolic Enzyme CHA1 Define a Novel Feedforward/Feedback Mechanism in the Response to Serine Availability*

    PubMed Central

    Montefusco, David J.; Newcomb, Benjamin; Gandy, Jason L.; Brice, Sarah E.; Matmati, Nabil; Cowart, L. Ashley; Hannun, Yusuf A.

    2012-01-01

    Targets of bioactive sphingolipids in Saccharomyces cerevisiae were previously identified using microarray experiments focused on sphingolipid-dependent responses to heat stress. One of these heat-induced genes is the serine deamidase/dehydratase Cha1 known to be regulated by increased serine availability. This study investigated the hypothesis that sphingolipids may mediate the induction of Cha1 in response to serine availability. The results showed that inhibition of de novo synthesis of sphingolipids, pharmacologically or genetically, prevented the induction of Cha1 in response to increased serine availability. Additional studies implicated the sphingoid bases phytosphingosine and dihydrosphingosine as the likely mediators of Cha1 up-regulation. The yeast protein kinases Pkh1 and Pkh2, known sphingoid base effectors, were found to mediate CHA1 up-regulation via the transcription factor Cha4. Because the results disclosed a role for sphingolipids in negative feedback regulation of serine metabolism, we investigated the effects of disrupting this mechanism on sphingolipid levels and on cell growth. Intriguingly, exposure of the cha1Δ strain to high serine resulted in hyperaccumulation of endogenous serine and in turn a significant accumulation of sphingoid bases and ceramides. Under these conditions, the cha1Δ strain displayed a significant growth defect that was sphingolipid-dependent. Together, this work reveals a feedforward/feedback loop whereby the sphingoid bases serve as sensors of serine availability and mediate up-regulation of Cha1 in response to serine availability, which in turn regulates sphingolipid levels by limiting serine accumulation. PMID:22277656

  16. Sphingosine-1-Phosphate Lyase Deficient Cells as a Tool to Study Protein Lipid Interactions

    PubMed Central

    Gerl, Mathias J.; Bittl, Verena; Kirchner, Susanne; Sachsenheimer, Timo; Brunner, Hanna L.; Lüchtenborg, Christian; Özbalci, Cagakan; Wiedemann, Hannah; Wegehingel, Sabine; Nickel, Walter; Haberkant, Per; Schultz, Carsten; Krüger, Marcus; Brügger, Britta

    2016-01-01

    Cell membranes contain hundreds to thousands of individual lipid species that are of structural importance but also specifically interact with proteins. Due to their highly controlled synthesis and role in signaling events sphingolipids are an intensely studied class of lipids. In order to investigate their metabolism and to study proteins interacting with sphingolipids, metabolic labeling based on photoactivatable sphingoid bases is the most straightforward approach. In order to monitor protein-lipid-crosslink products, sphingosine derivatives containing a reporter moiety, such as a radiolabel or a clickable group, are used. In normal cells, degradation of sphingoid bases via action of the checkpoint enzyme sphingosine-1-phosphate lyase occurs at position C2-C3 of the sphingoid base and channels the resulting hexadecenal into the glycerolipid biosynthesis pathway. In case the functionalized sphingosine looses the reporter moiety during its degradation, specificity towards sphingolipid labeling is maintained. In case degradation of a sphingosine derivative does not remove either the photoactivatable or reporter group from the resulting hexadecenal, specificity towards sphingolipid labeling can be achieved by blocking sphingosine-1-phosphate lyase activity and thus preventing sphingosine derivatives to be channeled into the sphingolipid-to-glycerolipid metabolic pathway. Here we report an approach using clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated nuclease Cas9 to create a sphingosine-1-phosphate lyase (SGPL1) HeLa knockout cell line to disrupt the sphingolipid-to-glycerolipid metabolic pathway. We found that the lipid and protein compositions as well as sphingolipid metabolism of SGPL1 knock-out HeLa cells only show little adaptations, which validates these cells as model systems to study transient protein-sphingolipid interactions. PMID:27100999

  17. Sphingosine-1-Phosphate Lyase Deficient Cells as a Tool to Study Protein Lipid Interactions.

    PubMed

    Gerl, Mathias J; Bittl, Verena; Kirchner, Susanne; Sachsenheimer, Timo; Brunner, Hanna L; Lüchtenborg, Christian; Özbalci, Cagakan; Wiedemann, Hannah; Wegehingel, Sabine; Nickel, Walter; Haberkant, Per; Schultz, Carsten; Krüger, Marcus; Brügger, Britta

    2016-01-01

    Cell membranes contain hundreds to thousands of individual lipid species that are of structural importance but also specifically interact with proteins. Due to their highly controlled synthesis and role in signaling events sphingolipids are an intensely studied class of lipids. In order to investigate their metabolism and to study proteins interacting with sphingolipids, metabolic labeling based on photoactivatable sphingoid bases is the most straightforward approach. In order to monitor protein-lipid-crosslink products, sphingosine derivatives containing a reporter moiety, such as a radiolabel or a clickable group, are used. In normal cells, degradation of sphingoid bases via action of the checkpoint enzyme sphingosine-1-phosphate lyase occurs at position C2-C3 of the sphingoid base and channels the resulting hexadecenal into the glycerolipid biosynthesis pathway. In case the functionalized sphingosine looses the reporter moiety during its degradation, specificity towards sphingolipid labeling is maintained. In case degradation of a sphingosine derivative does not remove either the photoactivatable or reporter group from the resulting hexadecenal, specificity towards sphingolipid labeling can be achieved by blocking sphingosine-1-phosphate lyase activity and thus preventing sphingosine derivatives to be channeled into the sphingolipid-to-glycerolipid metabolic pathway. Here we report an approach using clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated nuclease Cas9 to create a sphingosine-1-phosphate lyase (SGPL1) HeLa knockout cell line to disrupt the sphingolipid-to-glycerolipid metabolic pathway. We found that the lipid and protein compositions as well as sphingolipid metabolism of SGPL1 knock-out HeLa cells only show little adaptations, which validates these cells as model systems to study transient protein-sphingolipid interactions.

  18. Oral glucosylceramide reduces 2,4-dinitrofluorobenzene induced inflammatory response in mice by reducing TNF-alpha levels and leukocyte infiltration.

    PubMed

    Duan, Jingjing; Sugawara, Tatsuya; Sakai, Shota; Aida, Kazuhiko; Hirata, Takashi

    2011-06-01

    Sphingolipids are constituents of cellular membranes and play important roles as second messengers mediating cell functions. As significant components in foods, sphingolipids have been proven to be critical for human health. Moreover, diverse metabolic intermediates of sphingolipids are known to play key roles both in proinflammatory and in anti-inflammatory effects. However, the effect of dietary sphingolipids on inflammation is a complicated field that needs to be further assessed. Our study evaluated the effects of orally administered maize glucosylceramide (GluCer), one of the most conventional dietary sphingolipids, on inflammation using the 2,4-dinitro-1-fluorobenzene-treated BALB/c murine model. Oral administration of GluCer inhibited ear swelling and leukocyte infiltration to the inflammatory site, suggesting that dietary GluCer has anti-inflammatory properties. ELISA analyses revealed that oral administration of GluCer for 6 days had not modified the Th1/Th2 balance, but significantly down-regulated the activation of TNF-α at the inflammatory site. Based on these results, the down-regulation of TNF-α by dietary GluCer may suppress vascular permeability and reduce the migration of inflammatory cells. Our findings increase understanding of the actions of dietary sphingolipids on the balance of the immune response.

  19. Dynamic compositional changes of detergent-resistant plasma membrane microdomains during plant cold acclimation.

    PubMed

    Minami, Anzu; Furuto, Akari; Uemura, Matsuo

    2010-09-01

    Plants increase their freezing tolerance upon exposure to low, non-freezing temperatures, which is known as cold acclimation. Cold acclimation results in a decrease in the proportion of sphingolipids in the plasma membrane in many plants including Arabidopsis thaliana. The decrease in sphingolipids has been considered to contribute to the increase in the cryostability of the plasma membrane through regulating membrane fluidity. Recently we have proposed a possibility of another important sphingolipid function associated with cold acclimation. In animal cells, it has been known that the plasma membrane contains microdomains due to the chanracteristics of sphingolipids and sterols, and the sphingolipid- and sterol-enriched microdomains are thought to function as platforms for cell signaling, membrane trafficking and pathogen response. In our research on characterization of microdomain-associated lipids and proteins in Arabidopsis, cold-acclimation-induced decrease in sphingolipids resulted in a decrease of microdomains in the plasma membrane and there were considerable changes in membrane transport-, cytoskeleton- and endocytosis-related proteins in the microdomains during cold acclimation. Based on these results, we discuss a functional relationship between the changes in microdomain components and plant cold acclimation.

  20. LC-MS Based Sphingolipidomic Study on A2780 Human Ovarian Cancer Cell Line and its Taxol-resistant Strain

    PubMed Central

    Huang, Hao; Tong, Tian-Tian; Yau, Lee-Fong; Chen, Cheng-Yu; Mi, Jia-Ning; Wang, Jing-Rong; Jiang, Zhi-Hong

    2016-01-01

    Drug resistance elicited by cancer cells continue to cause huge problems world-wide, for example, tens of thousands of patients are suffering from taxol-resistant human ovarian cancer. However, its biochemical mechanisms remain unclear. Sphingolipid metabolic dysregulation has been increasingly regarded as one of the drug-resistant mechanisms for various cancers, which in turn provides potential targets for overcoming the resistance. In the current study, a well-established LC-MS based sphingolipidomic approach was applied to investigate the sphingolipid metabolism of A2780 and taxol-resistant A2780 (A2780T) human ovarian cancer cell lines. 102 sphingolipids (SPLs) were identified based on accurate mass and characteristic fragment ions, among which 12 species have not been reported previously. 89 were further quantitatively analyzed by using multiple reaction monitoring technique. Multivariate analysis revealed that the levels of 52 sphingolipids significantly altered in A2780T cells comparing to those of A2780 cells. These alterations revealed an overall increase of sphingomyelin levels and significant decrease of ceramides, hexosylceramides and lactosylceramides, which concomitantly indicated a deviated SPL metabolism in A2780T. This is the most comprehensive sphingolipidomic analysis of A2780 and A2780T, which investigated significantly changed sphingolipid profile in taxol-resistant cancer cells. The aberrant sphingolipid metabolism in A2780T could be one of the mechanisms of taxol-resistance. PMID:27703266

  1. Variable substrate preference among phospholipase D toxins from sicariid spiders

    DOE PAGES

    Lajoie, Daniel M.; Roberts, Sue A.; Zobel-Thropp, Pamela A.; ...

    2015-03-09

    Venoms of the sicariid spiders contain phospholipase D enzyme toxins that can cause severe dermonecrosis and even death in humans. These enzymes convert sphingolipid and lysolipid substrates to cyclic phosphates by activating a hydroxyl nucleophile present in both classes of lipid. The most medically relevant substrates are thought to be sphingomyelin and/or lysophosphatidylcholine. To better understand the substrate preference of these toxins, we used 31P NMR to compare the activity of three related but phylogenetically diverse sicariid toxins against a diverse panel of sphingolipid and lysolipid substrates. Two of the three showed significantly faster turnover of sphingolipids over lysolipids, andmore » all three showed a strong preference for positively charged (choline and/or ethanolamine) over neutral (glycerol and serine) headgroups. Strikingly, however, the enzymes vary widely in their preference for choline, the headgroup of both sphingomyelin and lysophosphatidylcholine, versus ethanolamine. An enzyme from Sicarius terrosus showed a strong preference for ethanolamine over choline, whereas two paralogous enzymes from Loxosceles arizonica either preferred choline or showed no significant preference. Intrigued by the novel substrate preference of the Sicarius enzyme, we solved its crystal structure at 2.1 Å resolution. Lastly, the evolution of variable substrate specificity may help explain the reduced dermonecrotic potential of some natural toxin variants, because mammalian sphingolipids use primarily choline as a positively charged headgroup; it may also be relevant for sicariid predatory behavior, because ethanolamine-containing sphingolipids are common in insect prey.« less

  2. Dual Functions of the Trans-2-Enoyl-CoA Reductase TER in the Sphingosine 1-Phosphate Metabolic Pathway and in Fatty Acid Elongation*

    PubMed Central

    Wakashima, Takeshi; Abe, Kensuke; Kihara, Akio

    2014-01-01

    The sphingolipid metabolite sphingosine 1-phosphate (S1P) functions as a lipid mediator and as a key intermediate of the sole sphingolipid to glycerophospholipid metabolic pathway (S1P metabolic pathway). In this pathway, S1P is converted to palmitoyl-CoA through 4 reactions, then incorporated mainly into glycerophospholipids. Although most of the genes responsible for the S1P metabolic pathway have been identified, the gene encoding the trans-2-enoyl-CoA reductase, responsible for the saturation step (conversion of trans-2-hexadecenoyl-CoA to palmitoyl-CoA) remains unidentified. In the present study, we show that TER is the missing gene in mammals using analyses involving yeast cells, deleting the TER homolog TSC13, and TER-knockdown HeLa cells. TER is known to be involved in the production of very long-chain fatty acids (VLCFAs). A significant proportion of the saturated and monounsaturated VLCFAs are used for sphingolipid synthesis. Therefore, TER is involved in both the production of VLCFAs used in the fatty acid moiety of sphingolipids as well as in the degradation of the sphingosine moiety of sphingolipids via S1P. PMID:25049234

  3. Microsomal Triglyceride Transfer Protein Transfers and Determines Plasma Concentrations of Ceramide and Sphingomyelin but Not Glycosylceramide*

    PubMed Central

    Iqbal, Jahangir; Walsh, Meghan T.; Hammad, Samar M.; Cuchel, Marina; Tarugi, Patrizia; Hegele, Robert A.; Davidson, Nicholas O.; Rader, Daniel J.; Klein, Richard L.; Hussain, M. Mahmood

    2015-01-01

    Sphingolipids, a large family of bioactive lipids, are implicated in stress responses, differentiation, proliferation, apoptosis, and other physiological processes. Aberrant plasma levels of sphingolipids contribute to metabolic disease, atherosclerosis, and insulin resistance. They are fairly evenly distributed in high density and apoB-containing lipoproteins (B-lps). Mechanisms involved in the transport of sphingolipids to the plasma are unknown. Here, we investigated the role of microsomal triglyceride transfer protein (MTP), required for B-lp assembly and secretion, in sphingolipid transport to the plasma. Abetalipoproteinemia patients with deleterious mutations in MTP and absence of B-lps had significantly lower plasma ceramide and sphingomyelin but normal hexosylceramide, lactosylceramide, and different sphingosines compared with unaffected controls. Furthermore, similar differential effects on plasma sphingolipids were seen in liver- and intestine-specific MTP knock-out (L,I-Mttp−/−) mice, suggesting that MTP specifically plays a role in the regulation of plasma ceramide and sphingomyelin. We hypothesized that MTP deficiency may affect either their synthesis or secretion. MTP deficiency had no effect on ceramide and sphingomyelin synthesis but reduced secretion from primary hepatocytes and hepatoma cells. Therefore, MTP is involved in ceramide and sphingomyelin secretion but not in their synthesis. We also found that MTP transferred these lipids between vesicles in vitro. Therefore, we propose that MTP might regulate plasma ceramide and sphingomyelin levels by transferring these lipids to B-lps in the liver and intestine and facilitating their secretion. PMID:26350457

  4. Interferon-Beta Increases Plasma Ceramides of Specific Chain Length in Multiple Sclerosis Patients, Unlike Fingolimod or Natalizumab

    PubMed Central

    Ottenlinger, Florian M.; Mayer, Christoph A.; Ferreirós, Nerea; Schreiber, Yannick; Schwiebs, Anja; Schmidt, Katrin G.; Ackermann, Hanns; Pfeilschifter, Josef M.; Radeke, Heinfried H.

    2016-01-01

    Fingolimod is used for the treatment of multiple sclerosis (MS) and targets receptors for the bioactive sphingolipid sphingosine-1-phosphate (S1P). Whether fingolimod or other MS therapies conversely affect plasma concentrations of sphingolipids has, however, not yet been analyzed. Herein, we quantified 15 representative sphingolipid species by mass spectrometry in plasma from relapsing-remitting MS patients currently under fingolimod (n = 24), natalizumab (n = 16), or IFN-β (n = 18) treatment. Healthy controls (n = 21) and untreated MS patients (n = 11) served as control groups. IFN-ß treatment strongly increased plasma level of C16:0, C18:0, C20:0, and C24:1 ceramides compared to healthy controls, untreated patients, or patients receiving fingolimod or natalizumab medication. Natalizumab treatment increased plasma concentrations of both S1P and sphinganine-1-phosphate, whereas fingolimod treatment did not affect any of these lipids. Correlations of sphingolipids with the Expanded Disability Status Scale and other disease specific parameters revealed no systemic change of sphingolipids in MS, independent of the respective treatment regime. These results indicate type I interferon treatment to cause a strong and specific increase in ceramide level. If confirmed in larger cohorts, these data have implications for the efficacy and adverse effects of IFN-β. Moreover, quantification of ceramides soon after therapy initiation may help to identify therapy-responsive patients. PMID:27857690

  5. Adaptation of Skyline for Targeted Lipidomics.

    PubMed

    Peng, Bing; Ahrends, Robert

    2016-01-04

    In response to the urgent need for analysis software that is capable of handling data from targeted high-throughput lipidomics experiments, we here present a systematic workflow for the straightforward method design and analysis of selected reaction monitoring data in lipidomics based on lipid building blocks. Skyline is a powerful software primarily designed for proteomics applications where it is widely used. We adapted this tool to a "Plug and Play" system for lipid research. This extension offers the unique capability to assemble targeted mass spectrometry methods for complex lipids easily by making use of building blocks. With simple yet tailored modifications, targeted methods to analyze main lipid classes such as glycerophospholipids, sphingolipids, glycerolipids, cholesteryl-esters, and cholesterol can be quickly introduced into Skyline for easy application by end users without distinct bioinformatics skills. To illustrate the benefits of our novel strategy, we used Skyline to quantify sphingolipids in mesenchymal stem cells. We demonstrate a simple method building procedure for sphingolipids screening, collision energy optimization, and absolute quantification of sphingolipids. In total, 72 sphingolipids were identified and absolutely quantified at the fatty acid scan species level by utilizing Skyline for data interpretation and visualization.

  6. Adipocyte Ceramides Regulate Subcutaneous Adipose Browning, Inflammation, and Metabolism.

    PubMed

    Chaurasia, Bhagirath; Kaddai, Vincent Andre; Lancaster, Graeme Iain; Henstridge, Darren C; Sriram, Sandhya; Galam, Dwight Lark Anolin; Gopalan, Venkatesh; Prakash, K N Bhanu; Velan, S Sendhil; Bulchand, Sarada; Tsong, Teh Jing; Wang, Mei; Siddique, Monowarul Mobin; Yuguang, Guan; Sigmundsson, Kristmundur; Mellet, Natalie A; Weir, Jacquelyn M; Meikle, Peter J; Bin M Yassin, M Shabeer; Shabbir, Asim; Shayman, James A; Hirabayashi, Yoshio; Shiow, Sue-Anne Toh Ee; Sugii, Shigeki; Summers, Scott A

    2016-12-13

    Adipocytes package incoming fatty acids into triglycerides and other glycerolipids, with only a fraction spilling into a parallel biosynthetic pathway that produces sphingolipids. Herein, we demonstrate that subcutaneous adipose tissue of type 2 diabetics contains considerably more sphingolipids than non-diabetic, BMI-matched counterparts. Whole-body and adipose tissue-specific inhibition/deletion of serine palmitoyltransferase (Sptlc), the first enzyme in the sphingolipid biosynthesis cascade, in mice markedly altered adipose morphology and metabolism, particularly in subcutaneous adipose tissue. The reduction in adipose sphingolipids increased brown and beige/brite adipocyte numbers, mitochondrial activity, and insulin sensitivity. The manipulation also increased numbers of anti-inflammatory M2 macrophages in the adipose bed and induced secretion of insulin-sensitizing adipokines. By comparison, deletion of serine palmitoyltransferase from macrophages had no discernible effects on metabolic homeostasis or adipose function. These data indicate that newly synthesized adipocyte sphingolipids are nutrient signals that drive changes in the adipose phenotype to influence whole-body energy expenditure and nutrient metabolism. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Variable Substrate Preference among Phospholipase D Toxins from Sicariid Spiders.

    PubMed

    Lajoie, Daniel M; Roberts, Sue A; Zobel-Thropp, Pamela A; Delahaye, Jared L; Bandarian, Vahe; Binford, Greta J; Cordes, Matthew H J

    2015-04-24

    Venoms of the sicariid spiders contain phospholipase D enzyme toxins that can cause severe dermonecrosis and even death in humans. These enzymes convert sphingolipid and lysolipid substrates to cyclic phosphates by activating a hydroxyl nucleophile present in both classes of lipid. The most medically relevant substrates are thought to be sphingomyelin and/or lysophosphatidylcholine. To better understand the substrate preference of these toxins, we used (31)P NMR to compare the activity of three related but phylogenetically diverse sicariid toxins against a diverse panel of sphingolipid and lysolipid substrates. Two of the three showed significantly faster turnover of sphingolipids over lysolipids, and all three showed a strong preference for positively charged (choline and/or ethanolamine) over neutral (glycerol and serine) headgroups. Strikingly, however, the enzymes vary widely in their preference for choline, the headgroup of both sphingomyelin and lysophosphatidylcholine, versus ethanolamine. An enzyme from Sicarius terrosus showed a strong preference for ethanolamine over choline, whereas two paralogous enzymes from Loxosceles arizonica either preferred choline or showed no significant preference. Intrigued by the novel substrate preference of the Sicarius enzyme, we solved its crystal structure at 2.1 Å resolution. The evolution of variable substrate specificity may help explain the reduced dermonecrotic potential of some natural toxin variants, because mammalian sphingolipids use primarily choline as a positively charged headgroup; it may also be relevant for sicariid predatory behavior, because ethanolamine-containing sphingolipids are common in insect prey. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Variable Substrate Preference among Phospholipase D Toxins from Sicariid Spiders*

    PubMed Central

    Lajoie, Daniel M.; Roberts, Sue A.; Zobel-Thropp, Pamela A.; Delahaye, Jared L.; Bandarian, Vahe; Binford, Greta J.; Cordes, Matthew H. J.

    2015-01-01

    Venoms of the sicariid spiders contain phospholipase D enzyme toxins that can cause severe dermonecrosis and even death in humans. These enzymes convert sphingolipid and lysolipid substrates to cyclic phosphates by activating a hydroxyl nucleophile present in both classes of lipid. The most medically relevant substrates are thought to be sphingomyelin and/or lysophosphatidylcholine. To better understand the substrate preference of these toxins, we used 31P NMR to compare the activity of three related but phylogenetically diverse sicariid toxins against a diverse panel of sphingolipid and lysolipid substrates. Two of the three showed significantly faster turnover of sphingolipids over lysolipids, and all three showed a strong preference for positively charged (choline and/or ethanolamine) over neutral (glycerol and serine) headgroups. Strikingly, however, the enzymes vary widely in their preference for choline, the headgroup of both sphingomyelin and lysophosphatidylcholine, versus ethanolamine. An enzyme from Sicarius terrosus showed a strong preference for ethanolamine over choline, whereas two paralogous enzymes from Loxosceles arizonica either preferred choline or showed no significant preference. Intrigued by the novel substrate preference of the Sicarius enzyme, we solved its crystal structure at 2.1 Å resolution. The evolution of variable substrate specificity may help explain the reduced dermonecrotic potential of some natural toxin variants, because mammalian sphingolipids use primarily choline as a positively charged headgroup; it may also be relevant for sicariid predatory behavior, because ethanolamine-containing sphingolipids are common in insect prey. PMID:25752604

  9. Plasma C16-Cer levels are increased in patients with preterm labor.

    PubMed

    Laudanski, Piotr; Charkiewicz, Karol; Kisielewski, Rafal; Kuc, Pawel; Koc-Zorawska, Ewa; Raba, Grzegorz; Kraczkowski, Janusz; Dymicka-Piekarska, Violetta; Chabowski, Adrian; Kacerovsky, Marian; Jacobsson, Bo; Zabielski, Piotr; Blachnio-Zabielska, Agnieszka

    2016-03-01

    The pathogenesis of preterm labor is fragmentarily explained. The most widely accepted theory points out to infection and inflammation as possible causes, which can be mediated by potentially different factors, including sphingolipid mediators. Sphingolipids are a class of lipids that have been shown as important mediators in various cell processes such as: proliferation, growth, apoptosis, stress response, necrosis and inflammation. The aim of the study was to assess plasma concentrations of selected sphingolipids in patients with preterm labor. We used ultra-high performance liquid chromatography with triple mass spectrometry (UHPLC-ESI-MS/MS) to assess plasma concentrations of the 11 sphingolipids in patients presenting with symptoms of preterm labor (n=61) and threatened preterm labor (n=40). We observed a statistically significant increase (p-value<0.004) in plasma concentrations of C16-Cer in patients with preterm labor as compared to the control group. We also found C16-Cer to be the best predictor of preterm labor in the group of patients with symptoms occurring after 32 weeks of gestation. Our findings show a possible involvement of selected sphingolipids, especially C16-Cer, in the pathogenesis of preterm labor. Their role as predictors of preterm delivery needs to be validated in the future on larger group of patients. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Revealing a signaling role of phytosphingosine-1-phosphate in yeast.

    PubMed

    Cowart, L Ashley; Shotwell, Matthew; Worley, Mitchell L; Richards, Adam J; Montefusco, David J; Hannun, Yusuf A; Lu, Xinghua

    2010-01-01

    Sphingolipids including sphingosine-1-phosphate and ceramide participate in numerous cell programs through signaling mechanisms. This class of lipids has important functions in stress responses; however, determining which sphingolipid mediates specific events has remained encumbered by the numerous metabolic interconnections of sphingolipids, such that modulating a specific lipid of interest through manipulating metabolic enzymes causes 'ripple effects', which change levels of many other lipids. Here, we develop a method of integrative analysis for genomic, transcriptomic, and lipidomic data to address this previously intractable problem. This method revealed a specific signaling role for phytosphingosine-1-phosphate, a lipid with no previously defined specific function in yeast, in regulating genes required for mitochondrial respiration through the HAP complex transcription factor. This approach could be applied to extract meaningful biological information from a similar experimental design that produces multiple sets of high-throughput data.

  11. Revealing a signaling role of phytosphingosine-1-phosphate in yeast

    PubMed Central

    Cowart, L Ashley; Shotwell, Matthew; Worley, Mitchell L; Richards, Adam J; Montefusco, David J; Hannun, Yusuf A; Lu, Xinghua

    2010-01-01

    Sphingolipids including sphingosine-1-phosphate and ceramide participate in numerous cell programs through signaling mechanisms. This class of lipids has important functions in stress responses; however, determining which sphingolipid mediates specific events has remained encumbered by the numerous metabolic interconnections of sphingolipids, such that modulating a specific lipid of interest through manipulating metabolic enzymes causes ‘ripple effects', which change levels of many other lipids. Here, we develop a method of integrative analysis for genomic, transcriptomic, and lipidomic data to address this previously intractable problem. This method revealed a specific signaling role for phytosphingosine-1-phosphate, a lipid with no previously defined specific function in yeast, in regulating genes required for mitochondrial respiration through the HAP complex transcription factor. This approach could be applied to extract meaningful biological information from a similar experimental design that produces multiple sets of high-throughput data. PMID:20160710

  12. Plasma membrane organization and function: moving past lipid rafts.

    PubMed

    Kraft, Mary L

    2013-09-01

    "Lipid raft" is the name given to the tiny, dynamic, and ordered domains of cholesterol and sphingolipids that are hypothesized to exist in the plasma membranes of eukaryotic cells. According to the lipid raft hypothesis, these cholesterol- and sphingolipid-enriched domains modulate the protein-protein interactions that are essential for cellular function. Indeed, many studies have shown that cellular levels of cholesterol and sphingolipids influence plasma membrane organization, cell signaling, and other important biological processes. Despite 15 years of research and the application of highly advanced imaging techniques, data that unambiguously demonstrate the existence of lipid rafts in mammalian cells are still lacking. This Perspective summarizes the results that challenge the lipid raft hypothesis and discusses alternative hypothetical models of plasma membrane organization and lipid-mediated cellular function.

  13. Sphingolipidomics: An Important Mechanistic Tool for Studying Fungal Pathogens

    PubMed Central

    Singh, Ashutosh; Del Poeta, Maurizio

    2016-01-01

    Sphingolipids form of a unique and complex group of bioactive lipids in fungi. Structurally, sphingolipids of fungi are quite diverse with unique differences in the sphingoid backbone, amide linked fatty acyl chain and the polar head group. Two of the most studied and conserved sphingolipid classes in fungi are the glucosyl- or galactosyl-ceramides and the phosphorylinositol containing phytoceramides. Comprehensive structural characterization and quantification of these lipids is largely based on advanced analytical mass spectrometry based lipidomic methods. While separation of complex lipid mixtures is achieved through high performance liquid chromatography, the soft – electrospray ionization tandem mass spectrometry allows a high sensitivity and selectivity of detection. Herein, we present an overview of lipid extraction, chromatographic separation and mass spectrometry employed in qualitative and quantitative sphingolipidomics in fungi. PMID:27148190

  14. Dihydroceramides: From Bit Players to Lead Actors*

    PubMed Central

    Siddique, Monowarul Mobin; Li, Ying; Chaurasia, Bhagirath; Kaddai, Vincent A.; Summers, Scott A.

    2015-01-01

    Sphingolipid synthesis involves a highly conserved biosynthetic pathway that produces fundamental precursors of complex sphingolipids. The final reaction involves the insertion of a double bond into dihydroceramides to generate the more abundant ceramides, which are converted to sphingomyelins and glucosylceramides/gangliosides by the addition of polar head groups. Although ceramides have long been known to mediate cellular stress responses, the dihydroceramides that are transiently produced during de novo sphingolipid synthesis were deemed inert. Evidence published in the last few years suggests that these dihydroceramides accumulate to a far greater extent in tissues than previously thought. Moreover, they have biological functions that are distinct and non-overlapping with those of the more prevalent ceramides. Roles are being uncovered in autophagy, hypoxia, and cellular proliferation, and the lipids are now implicated in the etiology, treatment, and/or diagnosis of diabetes, cancer, ischemia/reperfusion injury, and neurodegenerative diseases. This minireview summarizes recent findings on this emerging class of bioactive lipids. PMID:25947377

  15. Dihydroceramide desaturase 1, the gatekeeper of ceramide induced lipotoxicity.

    PubMed

    Rodriguez-Cuenca, S; Barbarroja, N; Vidal-Puig, A

    2015-01-01

    The pathogenic relevance of sphingolipid metabolism is increasingly being recognised. Here we elaborate on a new player within the sphingolipid field: the degs1 enzyme, a recently discovered enzyme that catalyses the final step in the de novo biosynthesis of ceramides controlling the step from dihydroceramides to ceramides. Here, we describe its function and dysregulation by factors such as oxidative stress, hypoxia and inflammation and provide evidence indicating that dihydroceramides constitute a biologically active molecule from the sphingolipid family with certain differential characteristics with respect to its delta-4 unsaturated counterparts, the ceramides. Finally we present pathophysiological scenarios characterised by specific increases in dihydroceramide that challenge the concept that "all ceramides species are the same". This article is part of a Special Issue entitled Linking transcription to physiology in lipodomics. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. Sphingomyelin Synthase 1 Is Essential for Male Fertility in Mice.

    PubMed

    Wittmann, Anke; Grimm, Marcus O W; Scherthan, Harry; Horsch, Marion; Beckers, Johannes; Fuchs, Helmut; Gailus-Durner, Valerie; Hrabě de Angelis, Martin; Ford, Steven J; Burton, Neal C; Razansky, Daniel; Trümbach, Dietrich; Aichler, Michaela; Walch, Axel Karl; Calzada-Wack, Julia; Neff, Frauke; Wurst, Wolfgang; Hartmann, Tobias; Floss, Thomas

    2016-01-01

    Sphingolipids and the derived gangliosides have critical functions in spermatogenesis, thus mutations in genes involved in sphingolipid biogenesis are often associated with male infertility. We have generated a transgenic mouse line carrying an insertion in the sphingomyelin synthase gene Sms1, the enzyme which generates sphingomyelin species in the Golgi apparatus. We describe the spermatogenesis defect of Sms1-/- mice, which is characterized by sloughing of spermatocytes and spermatids, causing progressive infertility of male homozygotes. Lipid profiling revealed a reduction in several long chain unsaturated phosphatidylcholins, lysophosphatidylcholins and sphingolipids in the testes of mutants. Multi-Spectral Optoacoustic Tomography indicated blood-testis barrier dysfunction. A supplementary diet of the essential omega-3 docosahexaenoic acid and eicosapentaenoic acid diminished germ cell sloughing from the seminiferous epithelium and restored spermatogenesis and fertility in 50% of previously infertile mutants. Our findings indicate that SMS1 has a wider than anticipated role in testis polyunsaturated fatty acid homeostasis and for male fertility.

  17. Sphingomyelin Synthase 1 Is Essential for Male Fertility in Mice

    PubMed Central

    Scherthan, Harry; Horsch, Marion; Beckers, Johannes; Fuchs, Helmut; Gailus-Durner, Valerie; Hrabě de Angelis, Martin; Ford, Steven J.; Burton, Neal C.; Razansky, Daniel; Trümbach, Dietrich; Aichler, Michaela; Walch, Axel Karl; Calzada-Wack, Julia; Neff, Frauke; Wurst, Wolfgang; Hartmann, Tobias; Floss, Thomas

    2016-01-01

    Sphingolipids and the derived gangliosides have critical functions in spermatogenesis, thus mutations in genes involved in sphingolipid biogenesis are often associated with male infertility. We have generated a transgenic mouse line carrying an insertion in the sphingomyelin synthase gene Sms1, the enzyme which generates sphingomyelin species in the Golgi apparatus. We describe the spermatogenesis defect of Sms1-/- mice, which is characterized by sloughing of spermatocytes and spermatids, causing progressive infertility of male homozygotes. Lipid profiling revealed a reduction in several long chain unsaturated phosphatidylcholins, lysophosphatidylcholins and sphingolipids in the testes of mutants. Multi-Spectral Optoacoustic Tomography indicated blood-testis barrier dysfunction. A supplementary diet of the essential omega-3 docosahexaenoic acid and eicosapentaenoic acid diminished germ cell sloughing from the seminiferous epithelium and restored spermatogenesis and fertility in 50% of previously infertile mutants. Our findings indicate that SMS1 has a wider than anticipated role in testis polyunsaturated fatty acid homeostasis and for male fertility. PMID:27788151

  18. Producing human ceramide-NS by metabolic engineering using yeast Saccharomyces cerevisiae

    PubMed Central

    Murakami, Suguru; Shimamoto, Toshi; Nagano, Hideaki; Tsuruno, Masahiro; Okuhara, Hiroaki; Hatanaka, Haruyo; Tojo, Hiromasa; Kodama, Yukiko; Funato, Kouichi

    2015-01-01

    Ceramide is one of the most important intercellular components responsible for the barrier and moisture retention functions of the skin. Because of the risks involved with using products of animal origin and the low productivity of plants, the availability of ceramides is currently limited. In this study, we successfully developed a system that produces sphingosine-containing human ceramide-NS in the yeast Saccharomyces cerevisiae by eliminating the genes for yeast sphingolipid hydroxylases (encoded by SUR2 and SCS7) and introducing the gene for a human sphingolipid desaturase (encoded by DES1). The inactivation of the ceramidase gene YDC1, overexpression of the inositol phosphosphingolipid phospholipase C gene ISC1, and endoplasmic reticulum localization of the DES1 gene product resulted in enhanced production of ceramide-NS. The engineered yeast strains can serve as hosts not only for providing a sustainable source of ceramide-NS but also for developing further systems to produce sphingosine-containing sphingolipids. PMID:26573460

  19. Producing human ceramide-NS by metabolic engineering using yeast Saccharomyces cerevisiae.

    PubMed

    Murakami, Suguru; Shimamoto, Toshi; Nagano, Hideaki; Tsuruno, Masahiro; Okuhara, Hiroaki; Hatanaka, Haruyo; Tojo, Hiromasa; Kodama, Yukiko; Funato, Kouichi

    2015-11-17

    Ceramide is one of the most important intercellular components responsible for the barrier and moisture retention functions of the skin. Because of the risks involved with using products of animal origin and the low productivity of plants, the availability of ceramides is currently limited. In this study, we successfully developed a system that produces sphingosine-containing human ceramide-NS in the yeast Saccharomyces cerevisiae by eliminating the genes for yeast sphingolipid hydroxylases (encoded by SUR2 and SCS7) and introducing the gene for a human sphingolipid desaturase (encoded by DES1). The inactivation of the ceramidase gene YDC1, overexpression of the inositol phosphosphingolipid phospholipase C gene ISC1, and endoplasmic reticulum localization of the DES1 gene product resulted in enhanced production of ceramide-NS. The engineered yeast strains can serve as hosts not only for providing a sustainable source of ceramide-NS but also for developing further systems to produce sphingosine-containing sphingolipids.

  20. Normal fur development and sebum production depends on fatty acid 2-hydroxylase expression in sebaceous glands.

    PubMed

    Maier, Helena; Meixner, Marion; Hartmann, Dieter; Sandhoff, Roger; Wang-Eckhardt, Lihua; Zöller, Inge; Gieselmann, Volkmar; Eckhardt, Matthias

    2011-07-22

    2-Hydroxylated fatty acid (HFA)-containing sphingolipids are abundant in mammalian skin and are believed to play a role in the formation of the epidermal barrier. Fatty acid 2-hydroxylase (FA2H), required for the synthesis of 2-hydroxylated sphingolipids in various organs, is highly expressed in skin, and previous in vitro studies demonstrated its role in the synthesis of HFA sphingolipids in human keratinocytes. Unexpectedly, however, mice deficient in FA2H did not show significant changes in their epidermal HFA sphingolipids. Expression of FA2H in murine skin was restricted to the sebaceous glands, where it was required for synthesis of 2-hydroxylated glucosylceramide and a fraction of type II wax diesters. Absence of FA2H resulted in hyperproliferation of sebocytes and enlarged sebaceous glands during hair follicle morphogenesis and anagen (active growth phase) in adult mice. This was accompanied by a significant up-regulation of the epidermal growth factor receptor ligand epigen in sebocytes. Loss of FA2H significantly altered the composition and physicochemical properties of sebum, which often blocked the hair canal, apparently causing a delay in the hair fiber exit. Furthermore, mice lacking FA2H displayed a cycling alopecia with hair loss in telogen. These results underline the importance of the sebaceous glands and suggest a role of specific sebaceous gland or sebum lipids, synthesized by FA2H, in the hair follicle homeostasis.

  1. Normal Fur Development and Sebum Production Depends on Fatty Acid 2-Hydroxylase Expression in Sebaceous Glands*

    PubMed Central

    Maier, Helena; Meixner, Marion; Hartmann, Dieter; Sandhoff, Roger; Wang-Eckhardt, Lihua; Zöller, Inge; Gieselmann, Volkmar; Eckhardt, Matthias

    2011-01-01

    2-Hydroxylated fatty acid (HFA)-containing sphingolipids are abundant in mammalian skin and are believed to play a role in the formation of the epidermal barrier. Fatty acid 2-hydroxylase (FA2H), required for the synthesis of 2-hydroxylated sphingolipids in various organs, is highly expressed in skin, and previous in vitro studies demonstrated its role in the synthesis of HFA sphingolipids in human keratinocytes. Unexpectedly, however, mice deficient in FA2H did not show significant changes in their epidermal HFA sphingolipids. Expression of FA2H in murine skin was restricted to the sebaceous glands, where it was required for synthesis of 2-hydroxylated glucosylceramide and a fraction of type II wax diesters. Absence of FA2H resulted in hyperproliferation of sebocytes and enlarged sebaceous glands during hair follicle morphogenesis and anagen (active growth phase) in adult mice. This was accompanied by a significant up-regulation of the epidermal growth factor receptor ligand epigen in sebocytes. Loss of FA2H significantly altered the composition and physicochemical properties of sebum, which often blocked the hair canal, apparently causing a delay in the hair fiber exit. Furthermore, mice lacking FA2H displayed a cycling alopecia with hair loss in telogen. These results underline the importance of the sebaceous glands and suggest a role of specific sebaceous gland or sebum lipids, synthesized by FA2H, in the hair follicle homeostasis. PMID:21628453

  2. Age‐related remodeling of small arteries is accompanied by increased sphingomyelinase activity and accumulation of long‐chain ceramides

    PubMed Central

    Ohanian, Jacqueline; Liao, Aiyin; Forman, Simon P.; Ohanian, Vasken

    2014-01-01

    Abstract The structure and function of large arteries alters with age leading to increased risk of cardiovascular disease. Age‐related large artery remodeling and arteriosclerosis is associated with increased collagen deposition, inflammation, and endothelial dysfunction. Bioactive sphingolipids are known to regulate these processes, and are also involved in aging and cellular senescence. However, less is known about age‐associated alterations in small artery morphology and function or whether changes in arterial sphingolipids occur in aging. We show that mesenteric small arteries from old sheep have increased lumen diameter and media thickness without a change in media to lumen ratio, indicative of outward hypertrophic remodeling. This remodeling occurred without overt changes in blood pressure or pulse pressure indicating it was a consequence of aging per se. There was no age‐associated change in mechanical properties of the arteries despite an increase in total collagen content and deposition of collagen in a thickened intima layer in arteries from old animals. Analysis of the sphingolipid profile showed an increase in long‐chain ceramide (C14–C20), but no change in the levels of sphingosine or sphingosine‐1‐phosphate in arteries from old compared to young animals. This was accompanied by a parallel increase in acid and neutral sphingomyelinase activity in old arteries compared to young. This study demonstrates remodeling of small arteries during aging that is accompanied by accumulation of long‐chain ceramides. This suggests that sphingolipids may be important mediators of vascular aging. PMID:24872355

  3. Circulating levels of sphingosine-1-phosphate are elevated in severe, but not mild psoriasis and are unresponsive to anti-TNF-α treatment

    NASA Astrophysics Data System (ADS)

    Checa, Antonio; Xu, Ning; Sar, Daniel G.; Haeggström, Jesper Z.; Ståhle, Mona; Wheelock, Craig E.

    2015-07-01

    Sphingolipids are bioactive molecules with a putative role in inflammation. Alterations in sphingolipids, in particular ceramides, have been consistently observed in psoriatic skin. Herein, we quantified the circulating sphingolipid profile in individuals with mild or severe psoriasis as well as healthy controls. In addition, the effects of anti-TNF-α treatment were determined. Levels of sphingoid bases, including sphingosine-1-phosphate (S1P), increased in severe (P < 0.001 n = 32), but not in mild (n = 32), psoriasis relative to healthy controls (n = 32). These alterations were not reversed in severe patients (n = 16) after anti-TNF-α treatment despite significant improvement in psoriasis lesions. Circulating levels of sphingomyelins and ceramides shifted in a fatty acid chain length-dependent manner. These alterations were also observed in psoriasis skin lesions and were associated with changes in mRNA levels of ceramide synthases. The lack of S1P response to treatment may have pathobiological implications due to its close relation to the vascular and immune systems. In particular, increased levels of sphingolipids and especially S1P in severe psoriasis patients requiring biological treatment may potentially be associated with cardiovascular comorbidities. The fact that shifts in S1P levels were not ameliorated by anti-TNF-α treatment, despite improvements in the skin lesions, further supports targeting S1P receptors as therapy for severe psoriasis.

  4. Finding pathway-modulating genes from a novel Ontology Fingerprint-derived gene network

    PubMed Central

    Qin, Tingting; Matmati, Nabil; Tsoi, Lam C.; Mohanty, Bidyut K.; Gao, Nan; Tang, Jijun; Lawson, Andrew B.; Hannun, Yusuf A.; Zheng, W. Jim

    2014-01-01

    To enhance our knowledge regarding biological pathway regulation, we took an integrated approach, using the biomedical literature, ontologies, network analyses and experimental investigation to infer novel genes that could modulate biological pathways. We first constructed a novel gene network via a pairwise comparison of all yeast genes’ Ontology Fingerprints—a set of Gene Ontology terms overrepresented in the PubMed abstracts linked to a gene along with those terms’ corresponding enrichment P-values. The network was further refined using a Bayesian hierarchical model to identify novel genes that could potentially influence the pathway activities. We applied this method to the sphingolipid pathway in yeast and found that many top-ranked genes indeed displayed altered sphingolipid pathway functions, initially measured by their sensitivity to myriocin, an inhibitor of de novo sphingolipid biosynthesis. Further experiments confirmed the modulation of the sphingolipid pathway by one of these genes, PFA4, encoding a palmitoyl transferase. Comparative analysis showed that few of these novel genes could be discovered by other existing methods. Our novel gene network provides a unique and comprehensive resource to study pathway modulations and systems biology in general. PMID:25063300

  5. Lipid mediators link cells progression with placental and neural tube defects after maternal fumonisin exposure

    USDA-ARS?s Scientific Manuscript database

    Fumonisin B1 (FB1) is a mycotoxin produced by a common fungal contaminant of maize. Increased risk for neural tube defects (NTDs) is observed in populations that rely on maize as a dietary staple. FB1 inhibits ceramide synthase, resulting in altered pools of biologically active sphingolipids. FB1...

  6. The Immunological Functions of Saposins

    PubMed Central

    Darmoise, Alexandre; Maschmeyer, Patrick; Winau, Florian

    2014-01-01

    Saposins or sphingolipid activator proteins (SAPs) are small, nonenzymatic glycoproteins that are ubiquitously present in lysosomes. SAPs comprise the five molecules saposins A–D and the GM2 activator protein. Saposins are essential for sphingolipid degradation and membrane digestion. On the one hand, they bind the respective hydrolases required to catabolize sphingolipid molecules; on the other hand, saposins can interact with intralysosomal membrane structures to render lipids accessible to their degrading enzymes. Thus, saposins bridge the physicochemical gap between lipid substrate and hydrophilic hydrolases. Accordingly, defects in saposin function can lead to lysosomal lipid accumulation. In addition to their specific functions in sphingolipid metabolism, saposins have membrane-perturbing properties. At the low pH of lysosomes, saposins get protonated and exhibit a high binding affinity for anionic phospholipids. Based on their universal principle to interact with membrane bilayers, we present the immunological functions of saposins with regard to lipid antigen presentation to CD1-restricted T cells, processing of apoptotic bodies for antigen delivery and cross-priming, as well as their potential antimicrobial impact. PMID:20510729

  7. Serum sphingolipidomic analyses reveal an upregulation of C16- ceramide and sphingosine-1-phosphate in hepatocellular carcinoma

    PubMed Central

    Grammatikos, Georgios; Schoell, Niklas; Ferreirós, Nerea; Bon, Dimitra; Herrmann, Eva; Farnik, Harald; Köberle, Verena; Piiper, Albrecht; Zeuzem, Stefan; Kronenberger, Bernd

    2016-01-01

    We have recently shown that major alterations of serum sphingolipid metabolites in chronic liver disease associate significantly with the stage of liver fibrosis in corresponding patients. In the current study we assessed via mass spectrometry serum concentrations of sphingolipid metabolites in a series of 122 patients with hepatocellular carcinoma (HCC) compared to an age- and sex-matched series of 127 patients with cirrhosis. We observed a highly significant upregulation of long and very long chain ceramides (C16-C24) in the serum of patients with HCC as compared to patients with cirrhosis (P < 0.001). Accordingly, dihydro-ceramides, synthetic precursors of ceramides and notably sphingosine, sphingosine-1-phosphate (S1P) and sphinganine-1-phosphate (SA1P) were upregulated in patients with HCC (P < 0.001). Especially the diagnostic accuracy of C16-ceramide and S1P, assessed by receiver operating curve (ROC) analysis, showed a higher area under the curve (AUC) value as compared to alpha fetoprotein (AFP) (0.999 and 0.985 versus 0.823, P < 0.001 respectively). In conclusion, serum levels of sphingolipid metabolites show a significant upregulation in patients with HCC as compared to patients with cirrhosis. Particularly C16-ceramide and S1P may serve as novel diagnostic markers for the identification of HCC in patients with liver diseases. Our data justify further investigations on the role of sphingolipids in HCC. PMID:26933996

  8. Autophagy Paradox and Ceramide

    PubMed Central

    Jiang, Wenhui; Ogretmen, Besim

    2013-01-01

    Sphingolipid molecules act as bioactive lipid messengers and exert their actions on the regulation of various cellular signaling pathways. Sphingolipids play essential roles in numerous cellular functions, including controlling cell inflammation, proliferation, death, migration, senescence, tumor metastasis and/or autophagy. Dysregulated sphingolipid metabolism has been also implicated in many human cancers. Macroatuophagy (referred to here as autophagy) “self-eating”, is characterized by nonselective sequestering of cytosolic materials by an isolation membrane, which can be either protective or lethal for cells. Ceramide (Cer), a central molecule of sphingolipid metabolism, has been extensively implicated in the control of autophagy. The increasing evidence suggests Cer is highly involved in mediating two opposing autophagic pathways, which regulate either cell survival or death, autophagy paradox. However, the underlying mechanism that regulates the autophagy paradox remains unclear. Therefore, this review focuses on recent studies with regard to the regulation of autophagy by Cer and elucidate the roles and mechanisms of action of Cer in controlling autophagy paradox. PMID:24055889

  9. Lipid mediators in the neural cell nucleus: their metabolism, signaling, and association with neurological disorders.

    PubMed

    Farooqui, Akhlaq A

    2009-08-01

    Lipid mediators are important endogenous regulators of neural cell proliferation, differentiation, oxidative stress, inflammation, and apoptosis. They originate from enzymic degradation of glycerophospholipids, sphingolipids, and cholesterol by phospholipases, sphingomyelinases, and cytochrome P450 hydroxylases, respectively. Arachidonic acid-derived lipid mediators are called eicosanoids. Eicosanoids have emerged as key regulators of cell proliferation, differentiation, oxidative stress, and neuroinflammation. Another arachidonic acid-derived lipid mediator is lipoxin. Eicosanoids have proinflammatory effects, whereas lipoxins produce antiinflammatory effects. The crossponding lipid mediators of docosahexaenoic acid metabolism are named docosanoids. They include resolvins, protectins, and neuroprotectins. Docosanoids produce antioxidant, anti-inflammatory, and antiapoptotic effects in the brain tissue. Other glycerophospholipid-derived lipid mediators are platelet-activating factor, lysophosphatidic acid, and endocannabinoids. Degradation of sphingolipids also results in the generation of sphingolipid-derived lipid mediators. Sphingolipid-derived lipid mediators are ceramide, ceramide 1-phosphate, sphingosine, and sphingosine 1-phosphate. They mediate cellular differentiation, cell growth, and apoptosis. Similarly, cholesterol-derived lipid mediators hydroxycholesterol and oxycholesterol produce apoptosis. Most of these mediators originate from the plasma membrane. The nucleus has its own set of enzymes and lipid mediators that originate from the nuclear envelope and matrix. The purpose of this commentary is to describe basic and clinical information on lipid mediators in the nucleus.

  10. Fumonisins, Tortillas and Neural Tube Defects: Untangling a Complex Issue

    USDA-ARS?s Scientific Manuscript database

    Fumonisin mycotoxins are found in corn and corn-based foods. Fumonisin B1 (FB1), the most common, disrupts sphingolipid metabolism thereby causing species-specific diseases in animals that include cancer in rodents and (birth) neural tube defects (NTD) in LM/Bc mice. Fumonisins’ affect on human heal...

  11. In vivo Metabolism of Hydrolyzed Fumonisin B1 and Fumonisin B1

    USDA-ARS?s Scientific Manuscript database

    Fumonisin B1 (FB1) is the most prevalent fumonisin mycotoxin found in corn and corn-based foods. It inhibits ceramide synthase, disrupts sphingolipid metabolism and function, is toxic to animals, causes cancer in rodents, and induces neural tube defects in some mouse strains. Its human health effect...

  12. Increased nuclear sphingoid base-1-phosphates and HDAC inhibition after fumonisin and FTY720-treatment: the link between epigenomic modifications and neural tube defects?

    USDA-ARS?s Scientific Manuscript database

    Introduction: Fumonisin B1 (FB1) is a mycotoxin produced by a common fungal contaminant of corn. Ingestion of FB1-contaminated food during early pregnancy is associated with increased risk for neural tube defects (NTDs). FB1 inhibits the enzyme ceramide synthase in de novo sphingolipid biosynthes...

  13. Reduction of Fumonisin Toxicity by Extrusion and Nixtamalization (Alkaline Cooking)

    USDA-ARS?s Scientific Manuscript database

    Fumonisins are found in corn. The most common, fumonisin FB1 (FB1) is toxic to animals, disrupts sphingolipid metabolism, and is a suspected risk factor for neural tube defects (NTDs; serious birth defect) and cancer in humans that consume contaminated corn as a diet staple. FB1 levels in foods an...

  14. Acid Sphingomyelinase Deficiency Prevents Diet-induced Hepatic Triacylglycerol Accumulation and Hyperglycemia in Mice*

    PubMed Central

    Deevska, Gergana M.; Rozenova, Krassimira A.; Giltiay, Natalia V.; Chambers, Melissa A.; White, James; Boyanovsky, Boris B.; Wei, Jia; Daugherty, Alan; Smart, Eric J.; Reid, Michael B.; Merrill, Alfred H.; Nikolova-Karakashian, Mariana

    2009-01-01

    Acid sphingomyelinase plays important roles in ceramide homeostasis, which has been proposed to be linked to insulin resistance. To test this association in vivo, acid sphingomyelinase deletion (asm–/–) was transferred to mice lacking the low density lipoprotein receptor (ldlr–/–), and then offsprings were placed on control or modified (enriched in saturated fat and cholesterol) diets for 10 weeks. The modified diet caused hypercholesterolemia in all genotypes; however, in contrast to asm+/+/ldlr–/–, the acid sphingomyelinase-deficient littermates did not display hepatic triacylglyceride accumulation, although sphingomyelin and other sphingolipids were substantially elevated, and the liver was enlarged. asm–/–/ldlr–/– mice on a modified diet did not accumulate body fat and were protected against diet-induced hyperglycemia and insulin resistance. Experiments with hepatocytes revealed that acid sphingomyelinase regulates the partitioning of the major fatty acid in the modified diet, palmitate, into two competitive and inversely related pools, triacylglycerides and sphingolipids, apparently via modulation of serine palmitoyltransferase, a rate-limiting enzyme in de novo sphingolipid synthesis. These studies provide evidence that acid sphingomyelinase activity plays an essential role in the regulation of glucose metabolism by regulating the hepatic accumulation of triacylglycerides and sphingolipids during consumption of a diet rich in saturated fats. PMID:19074137

  15. IDENTIFICATION OF FLOTILLIN-1 ON EIMERIA TENELLA SPOROZOITES AND ITS ROLE IN HOST CELL INVASION

    USDA-ARS?s Scientific Manuscript database

    Lipid rafts are detergent-resistant, liquid-ordered microdomains in plasma membranes that are enriched in cholesterol and sphingolipids and involved in intracellular signal transduction, membrane trafficking, and molecular sorting. In this study, we investigated the possibility that lipid rafts on E...

  16. Analytical methods in sphingolipidomics: Quantitative and profiling approaches in food analysis.

    PubMed

    Canela, Núria; Herrero, Pol; Mariné, Sílvia; Nadal, Pedro; Ras, Maria Rosa; Rodríguez, Miguel Ángel; Arola, Lluís

    2016-01-08

    In recent years, sphingolipidomics has emerged as an interesting omic science that encompasses the study of the full sphingolipidome characterization, content, structure and activity in cells, tissues or organisms. Like other omics, it has the potential to impact biomarker discovery, drug development and systems biology knowledge. Concretely, dietary food sphingolipids have gained considerable importance due to their extensively reported bioactivity. Because of the complexity of this lipid family and their diversity among foods, powerful analytical methodologies are needed for their study. The analytical tools developed in the past have been improved with the enormous advances made in recent years in mass spectrometry (MS) and chromatography, which allow the convenient and sensitive identification and quantitation of sphingolipid classes and form the basis of current sphingolipidomics methodologies. In addition, novel hyphenated nuclear magnetic resonance (NMR) strategies, new ionization strategies, and MS imaging are outlined as promising technologies to shape the future of sphingolipid analyses. This review traces the analytical methods of sphingolipidomics in food analysis concerning sample extraction, chromatographic separation, the identification and quantification of sphingolipids by MS and their structural elucidation by NMR. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Fumonisin as a possible contributing factor to neural tube defects in populations consuming large amounts of maize

    USDA-ARS?s Scientific Manuscript database

    Fumonisin B1 (FB) is an inhibitor of sphingolipid (SL) biosynthesis and folate transport and can induce neural tube defects (NTD) in mice. NTD incidence is high in countries where maize is a dietary staple and FB exposure is likely. In Guatemala the incidence of FB in maize has been well documented ...

  18. Comparative lipid analysis in the normal and cancerous organoids of MDCK cells

    PubMed Central

    Yoshizaki, Hisayoshi; Ogiso, Hideo; Okazaki, Toshiro; Kiyokawa, Etsuko

    2016-01-01

    Epithelial organs are made of a well-polarized monolayer of epithelial cells, and their morphology is maintained strictly for their proper functioning. The roles of lipids are not only to generate the membrane, but also to provide the specific domains for signal transduction, or to transmit signals as second messengers. By using a liquid chromatography-electrospray ionization mass spectrometry (LC-MS)/MS method, we here analyzed sphingolipids in MDCK cysts under various conditions. Our result showed that, compared to the three-dimensional cyst, the two-dimensional MDCK sheet is relatively enriched in sphingolipids. During cystogenesis, the contents of sphingomyelin (SM) and lactocylceramide (LacCer)—but, none those of ceramide, hexocylceramide, or GM3—are altered depending on their acyl chains. While the total SM is decreased more efficiently by SMS-1 knockdown than by SMS-2 knockdown, depletion of SMS-2, but not SMS-1, inhibits cyst growth. Finally upon the switching on of activated K-Ras expression which induces luminal cell filling, ceramide and LacCer are increased. Our parallel examinations of the microarray data for mRNA of sphingolipid metabolic enzymes failed to fully explain the remodelling of the sphingolipids of MDCK cysts. However, these results should be useful to investigate the cell-type- and structure-specific lipid metabolism. PMID:26783265

  19. Increased sphingoid base-1-phosphates and failure of neural tube closure after exposure to fumonisin or FTY720

    USDA-ARS?s Scientific Manuscript database

    Fumonisin B1 (FB1) is a mycotoxin produced by a common fungal contaminant of corn. Ingestion of FB1-contaminated food is associated with increased risk for neural tube defects (NTDs). FB1 induces NTDs in inbred LM/Bc mice. FB1 inhibits ceramide synthase in de novo sphingolipid biosynthesis, resultin...

  20. Accumulation of sphingoid bases and sphingoid base 1-phosphates: A possible mechanism for Fusarium verticillioides corn-seedling disease

    USDA-ARS?s Scientific Manuscript database

    Sphingolipids are important structural components of membranes involved in signaling pathways that regulate cell growth and death. Fumonisins (FB) are water soluble mycotoxins produced by F. verticillioides, which is parasitic to corn. FBs are inhibitors of ceramide synthase (CS), a key enzyme in sp...

  1. The Role of Sphingosine Kinase 2 in Apoptosis of Human Breast Cancer

    DTIC Science & Technology

    2005-05-01

    The sphingolipid metabolite sphingosine-1-phosphate ( S1P ) is the ligand for a family of five specific G protein-coupled receptors ( S1P sub 1-5) that...action is still a matter of debate. S1P is formed by the ATP-dependent phosphorylation of sphingosine catalyzed by types 1 and 2 sphingosine kinase

  2. Circulating levels of sphingosine-1-phosphate are elevated in severe, but not mild psoriasis and are unresponsive to anti-TNF-α treatment

    PubMed Central

    Checa, Antonio; Xu, Ning; Sar, Daniel G.; Haeggström, Jesper Z.; Ståhle, Mona; Wheelock, Craig E.

    2015-01-01

    Sphingolipids are bioactive molecules with a putative role in inflammation. Alterations in sphingolipids, in particular ceramides, have been consistently observed in psoriatic skin. Herein, we quantified the circulating sphingolipid profile in individuals with mild or severe psoriasis as well as healthy controls. In addition, the effects of anti-TNF-α treatment were determined. Levels of sphingoid bases, including sphingosine-1-phosphate (S1P), increased in severe (P < 0.001; n = 32), but not in mild (n = 32), psoriasis relative to healthy controls (n = 32). These alterations were not reversed in severe patients (n = 16) after anti-TNF-α treatment despite significant improvement in psoriasis lesions. Circulating levels of sphingomyelins and ceramides shifted in a fatty acid chain length-dependent manner. These alterations were also observed in psoriasis skin lesions and were associated with changes in mRNA levels of ceramide synthases. The lack of S1P response to treatment may have pathobiological implications due to its close relation to the vascular and immune systems. In particular, increased levels of sphingolipids and especially S1P in severe psoriasis patients requiring biological treatment may potentially be associated with cardiovascular comorbidities. The fact that shifts in S1P levels were not ameliorated by anti-TNF-α treatment, despite improvements in the skin lesions, further supports targeting S1P receptors as therapy for severe psoriasis. PMID:26174087

  3. Ablation of Ceramide Synthase 2 Causes Chronic Oxidative Stress Due to Disruption of the Mitochondrial Respiratory Chain*

    PubMed Central

    Zigdon, Hila; Kogot-Levin, Aviram; Park, Joo-Won; Goldschmidt, Ruth; Kelly, Samuel; Merrill, Alfred H.; Scherz, Avigdor; Pewzner-Jung, Yael; Saada, Ann; Futerman, Anthony H.

    2013-01-01

    Ceramide is a key intermediate in the pathway of sphingolipid biosynthesis and is an important intracellular messenger. We recently generated a ceramide synthase 2 (CerS2) null mouse that cannot synthesize very long acyl chain (C22-C24) ceramides. This mouse displays severe and progressive hepatopathy. Significant changes were observed in the sphingolipid profile of CerS2 null mouse liver, including elevated C16-ceramide and sphinganine levels in liver and in isolated mitochondrial fractions. Because ceramide may be involved in reactive oxygen species (ROS) formation, we examined whether ROS generation was affected in CerS2 null mice. Levels of a number of anti-oxidant enzymes were elevated, as were lipid peroxidation, protein nitrosylation, and ROS. ROS were generated from mitochondria due to impaired complex IV activity. C16-ceramide, sphingosine, and sphinganine directly inhibited complex IV activity in isolated mitochondria and in mitoplasts, whereas other ceramide species, sphingomyelin, and diacylglycerol were without effect. A fluorescent analog of sphinganine accumulated in mitochondria. Heart mitochondria did not display a substantial alteration in the sphingolipid profile or in complex IV activity. We suggest that C16-ceramide and/or sphinganine induce ROS formation through the modulation of mitochondrial complex IV activity, resulting in chronic oxidative stress. These results are of relevance for understanding modulation of ROS signaling by sphingolipids. PMID:23283968

  4. Finding pathway-modulating genes from a novel Ontology Fingerprint-derived gene network.

    PubMed

    Qin, Tingting; Matmati, Nabil; Tsoi, Lam C; Mohanty, Bidyut K; Gao, Nan; Tang, Jijun; Lawson, Andrew B; Hannun, Yusuf A; Zheng, W Jim

    2014-10-01

    To enhance our knowledge regarding biological pathway regulation, we took an integrated approach, using the biomedical literature, ontologies, network analyses and experimental investigation to infer novel genes that could modulate biological pathways. We first constructed a novel gene network via a pairwise comparison of all yeast genes' Ontology Fingerprints--a set of Gene Ontology terms overrepresented in the PubMed abstracts linked to a gene along with those terms' corresponding enrichment P-values. The network was further refined using a Bayesian hierarchical model to identify novel genes that could potentially influence the pathway activities. We applied this method to the sphingolipid pathway in yeast and found that many top-ranked genes indeed displayed altered sphingolipid pathway functions, initially measured by their sensitivity to myriocin, an inhibitor of de novo sphingolipid biosynthesis. Further experiments confirmed the modulation of the sphingolipid pathway by one of these genes, PFA4, encoding a palmitoyl transferase. Comparative analysis showed that few of these novel genes could be discovered by other existing methods. Our novel gene network provides a unique and comprehensive resource to study pathway modulations and systems biology in general.

  5. Change in activity of serine palmitoyltransferase affects sensitivity to syringomycin E in yeast Saccharomyces cerevisiae.

    PubMed

    Toume, Moeko; Tani, Motohiro

    2014-09-01

    Syringomycin E is a cyclic lipodepsipeptide produced by strains of the plant bacterium Pseudomonas syringae pv. syringae. Genetic studies involving the yeast Saccharomyces cerevisiae have revealed that complex sphingolipids play important roles in the action of syringomycin E. Here, we found a novel mutation that confers resistance to syringomycin E on yeast; that is, a deletion mutant of ORM1 and ORM2, which encode negative regulators of serine palmitoyltransferase catalyzing the initial step of sphingolipid biosynthesis, exhibited resistance to syringomycin E. On the contrary, overexpression of Orm2 resulted in high sensitivity to the toxin. Moreover, overexpression of Lcb1 and Lcb2, catalytic subunits of serine palmitoyltransferase, causes resistance to the toxin, whereas partial repression of expression of Lcb1 had the opposite effect. Partial reduction of complex sphingolipids by repression of expression of Aur1, an inositol phosphorylceramide synthase, also resulted in high sensitivity to the toxin. These results suggested that an increase in sphingolipid biosynthesis caused by a change in the activity of serine palmitoyltransferase causes resistance to syringomycin E.

  6. Spingolipids in the root play an important role in regulating the leaf ionome in Arabidopsis thaliana

    USDA-ARS?s Scientific Manuscript database

    Sphingolipid synthesis is initiated by condensation of serine with palmitoyl-CoA to produce 3-ketodihydrosphinganine (3-KDS), which is subsequently reduced by a 3-KDS reductase to dihydrosphinganine (DHS). Serine palmitoyltransferase was recently shown to be essential for plant viability, but the 3...

  7. Fingolimod for the treatment of neurological diseases—state of play and future perspectives

    PubMed Central

    Brunkhorst, Robert; Vutukuri, Rajkumar; Pfeilschifter, Waltraud

    2014-01-01

    Sphingolipids are a fascinating class of signaling molecules derived from the membrane lipid sphingomyelin. They show abundant expression in the brain. Complex sphingolipids such as glycosphingolipids (gangliosides and cerebrosides) regulate vesicular transport and lysosomal degradation and their dysregulation can lead to storage diseases with a neurological phenotype. More recently, simple sphingolipids such ceramide, sphingosine and sphingosine 1-phosphate (S1P) were discovered to signal in response to many extracellular stimuli. Forming an intricate signaling network, the balance of these readily interchangeable mediators is decisive for cell fate under stressful conditions. The immunomodulator fingolimod is the prodrug of an S1P receptor agonist. Following receptor activation, the drug leads to downregulation of the S1P1 receptor inducing functional antagonism. As the first drug to modulate the sphingolipid signaling pathway, it was marketed in 2010 for the treatment of multiple sclerosis (MS). At that time, immunomodulation was widely accepted as the key mechanism of fingolimod’s efficacy in MS. But given the excellent passage of this lipophilic compound into the brain and its massive brain accumulation as well as the abundant expression of S1P receptors on brain cells, it is conceivable that fingolimod also affects brain cells directly. Indeed, a seminal study showed that the protective effect of fingolimod in experimental autoimmune encephalitis (EAE), a murine MS model, is lost in mice lacking the S1P1 receptor on astrocytes, arguing for a specific role of astrocytic S1P signaling in MS. In this review, we discuss the role of sphingolipid mediators and their metabolizing enzymes in neurologic diseases and putative therapeutic strategies arising thereof. PMID:25309325

  8. Phytosphingosine as a specific inhibitor of growth and nutrient import in Saccharomyces cerevisiae.

    PubMed

    Chung, N; Mao, C; Heitman, J; Hannun, Y A; Obeid, L M

    2001-09-21

    In the yeast Saccharomyces cerevisiae, we have demonstrated a necessary role for sphingolipids in the heat stress response through inhibition of nutrient import (Chung, N., Jenkins, G. M., Hannun, Y. A., Heitman, J., and Obeid, L. M. (2000) J. Biol. Chem. 275, 17229-17232). In this study, we used a combination of pharmacological and genetic approaches to determine which endogenous sphingolipid is the likely mediator of growth inhibition. When cells were treated with exogenous phytosphingosine (PHS, 20 microm) or structurally similar or metabolically related molecules, including 3-ketodihydrosphingosine, dihydrosphingosine, C(2)-phytoceramide (PHC), and stearylamine, only PHS inhibited growth. Also, PHS was shown to inhibit uptake of uracil, tryptophan, leucine, and histidine. Again this effect was specific to PHS. Because of the dynamic nature of sphingolipid metabolism, however, it was difficult to conclude that growth inhibition was caused by PHS itself. By using mutant